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Antimicrobial peptide from mucus of Andrias davidianus: screening and purification by magnetic cell membrane separation technique
Accepted Manuscript Title: Antimicrobial peptide from mucus of Andrias davidianus: screening and purification by magnetic cell membrane separation technique Author: Jinjin Pei, Lei Jiang PII: DOI: Reference:
S0924-8579(17)30138-3 http://dx.doi.org/doi: 10.1016/j.ijantimicag.2017.02.013 ANTAGE 5093
To appear in:
International Journal of Antimicrobial Agents
Received date: Accepted date:
19-7-2016 10-2-2017
Please cite this article as: Jinjin Pei, Lei Jiang, Antimicrobial peptide from mucus of Andrias davidianus: screening and purification by magnetic cell membrane separation technique, International Journal of Antimicrobial Agents (2017), http://dx.doi.org/doi: 10.1016/j.ijantimicag.2017.02.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Antimicrobial peptide from mucus of Andrias davidianus: screening and purification by magnetic cell membrane separation technique
Jinjin Pei a,*, Lei Jiang b
a
Shaanxi Key Laboratory of Biology and Bioresources, Shaanxi University of
Technology, Chaoyang Road, Shaanxi University of Technology, Hanzhong, Shaanxi 723001, China b
Key Laboratory of Tibetan Medicine Research, Northwest Institute of Plateau
Biology, Chinese Academy of Sciences, Xining, Qinghai, China
ARTICLE INFO Article history: Received 19 July 2016 Accepted 10 February 2017
Keywords: Andrias davidianus Mucus Antimicrobial peptide Magnetic cell membrane separation
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Escherichia coli
* Corresponding author. Tel.: +86 916 264 1641. E-mail address: [email protected] (J. Pei).
Page 2 of 25
Highlights
An antimicrobial peptide (andricin 01) was isolated from the mucus of Andrias davidianus.
Andricin 01 was purified using an innovative magnetic cell membrane separation technique.
Andricin 01 is a novel antimicrobial peptide.
ABSTRACT Andrias davidianus, the Chinese giant salamander, has been used in traditional Chinese medicine for many decades. However, no antimicrobial peptides (AMPs) have been described from A. davidianus until now. Here we describe a novel AMP (andricin 01) isolated from the mucus of A. davidianus. The peptide was recovered using an innovative magnetic cell membrane separation technique and was characterised using mass spectrometry and circular dichroism (CD) spectroscopy. Andricin 01 is comprised of ten amino acid residues with a total molecular mass of 955.1 Da. CD spectrum analysis gave results similar to the archetypal random coil spectrum, consistent with the three-dimensional rendering calculated by current bioinformatics tools. Andricin 01 was found to be inhibitory both to Gram-negative and Gram-positive bacteria. Furthermore, the peptide at the minimal bacterial concentration did not show cell cytotoxicity against human hepatocytes or renal cells
Page 3 of 25
and did not show haemolytic activity against red blood cells, indicating that is potentially safe and effective for human use. Andricin 01 shows promise as a novel antibacterial that may provide an insight into the development of new drugs.
Page 4 of 25
1. Introduction In recent years, scientists have turned increasingly towards the study of plants and animals in the hope of identifying and characterising new antimicrobial peptides (AMPs) [1–5]. The reason for this is simple. Microbes reproduce and evolve at an exponential rate that is outpacing the discovery of new antimicrobial agents [4]. The emergence of strains of human pathogens that are resistant to most or all of the current lines of treatment [4,5] underpins the increasing need for better treatments [1–5].
Recent research has allowed us to elucidate the primary structure of >8000 AMPs (http://www.bicnirrh.res.in/antimicrobial) [4–10]. Amphibians comprise the oldest class of animals from which these compounds have been isolated [5–10]. Most of the environments that amphibians inhabit are damp and dark, favouring the survival of many pathogenic micro-organisms [6,7]. Accordingly, amphibians have adopted a complex system of AMPs in their immune system to defend against such attackers [8–10].
Andrias davidianus (Chinese giant salamander) is thought to have originated ca. 350 000 000 years ago [11]. Its skin secretes white mucus on stimulation; in regional folk medicine, this by-product is purported to reduce the symptoms and signs of dementia, apoplexy, tetanus and tuberculosis and to improve the outcome of various other
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disease states [12–14]. Until now, there has been little study of the potential bioactive peptides of salamander mucus. The purpose of this study was to evaluate potential antimicrobial components of A. davidianus mucus as potential treatments for human infections.
The methods employed to isolate peptides from heterogeneous mixtures frequently involve complex chromatographic procedures that are labour- and resource-intensive and are subject to failure [15–18]. More rapid and efficient methods to isolate and analyse AMPs are therefore required. Commonality in effective AMPs can and should be exploited [19,20]. For example, it is believed that all AMPs, regardless of their mechanism of action, require an interaction with the targeted bacterial cellular membrane for activity to be initiated [21–24]. Against this background, we aimed to rapidly screen for AMPs that are capable of interfacing with bacterial membranes using immobilised bacterial membrane liposome chromatography. Finally, peptide characteristics including structural parameters, activity spectra, haemolytic capability and cytotoxicity were investigated.
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2. Materials and methods 2.1. Preparation of magnetic nanoliposomes Ferromagnetic Fe3O4 nanoparticles were prepared by a hydrothermal method [25]. Lipid of Escherichia coli was extracted using 2:1 (v/v) chloroform:methanol 18]. Nanomagnetic liposome particles were prepared by the thin-film dispersion method [26].
2.2. Purification of antimicrobial peptides from mucus of Andrias davidianus Lyophilised powder of A. davidianus mucus was dissolved in phosphate-buffered saline (PBS) (pH 7.2, 10 mmol/L) with 50 mmol/L NaCl, was filtered using a cellulose membrane (Ultracel PLGC04310; Merck Millipore, Billerica, MA) with a cut-off molecular weight at 10 kDa and was then dialysed with a 100 Da membrane (SP131048; Yuanye Bio, Shanghai, China) to obtain the crude sample. Magnetic nanoliposomes were then added and were co-cultured at 37 C for 24 h. After collection of magnetic liposomes by a magnet column, PBS (pH 7.2, 10 mM) with 50 mM NaCl served as an isocratic mobile phase at 25 C. Absorbance was recorded at 215 nm using a UV/Vis DAD (Agilent 1260 series; Agilent Technologies, Santa Clara, CA). Peaks were detected and were analysed for their potential bactericidal effect against E. coli CICC 10300 by the agar diffusion method [27]. From this, the fraction that exhibited the highest activity was put through reverse-phase high-performance
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liquid chromatography (RP-HPLC) (Waters Symmetry C18 column, 250 4.6 mm, 5 m; Waters, Dublin, Ireland). A gradient method was used for separation (with mobile phase B, 5−100% in 40 min) at a flow rate of 0.5 mL/min at 30 ± 2 C. The two mobile phases used were: mobile phase (A) 0.05% (v/v) trifluoroacetic acid; and mobile phase (B) 100% acetonitrile.
2.3. Structural characterisation The purified AMP (andricin 01) was tested by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). The sequence was searched for similarity against the NCBI BLAST database (http://blast.ncbi.nlm.nih.gov/) as well as databases accessible through the Bioinformatics Resource Portal ExPASy (http://www.expasy.org). Measurements were taken using circular dichroism (CD) spectroscopy on a Jasco J-810 CD spectrometer (Jasco Co., Tokyo, Japan) with a 0.5 mm path length at UV 195–250 nm at Shaanxi Normal University (Xi'an, China). Molecular stimulation software HyperChem 8.0 (HyperCube Inc., Gainesville, FL) was used to predict the structure under the lowest energy state.
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2.4. Synthesis of peptides AMPs were synthesised by Beijing Huada Protein Engineering Ltd. (Beijing, China) [27]. MALDI-TOF/MS and RP-HPLC were used to confirm peptide purity and identity.
2.5. Activity of the antimicrobial peptide The activity of andricin 01 was tested against the indicator strains listed in Table 1. An increase in optical density at 600 nm was used to monitor growth. Poor nutrient broth and poor potato dextrose broth (0.5 PDB) were used to culture bacteria. The minimum inhibitory concentration (MIC), defined as the lowest concentration of AMP needed to inhibit growth, was observed following incubation at 30 C for 18 h [27], and the minimum bacterial concentration (MBC) was defined as the lowest AMP concentration of AMP with no visual growth observed after 96 h of incubation at 30 C. Tests were conducted three times.
2.6. Safety evaluation The cytotoxicity of andricin 01 was evaluated by the MTT cell viability assay with a human hepatocyte cell line (HL-7702) and human renal epithelial cells (HREpiC) (ATCC, Manassas, VA) [28,29]. Percent haemolysis was determined with respect to a lysis control (red blood cells in 0.1% Triton X-100) and a negative control (in PBS). Assays were conducted in triplicate [18].
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2.7. Statistics MALDI-TOF/MS results were interpreted using Mass Lynx 4.1 software (Waters, Milford, MA) to calculate accurate molecular masses [4]. SPSS Statistics v.18.0 (SPSS Inc., Chicago, IL) was used for data analysis [18]. Data were averaged across replicates. Results are reported as the mean ± standard deviation.
3. Results 3.1. Purification of andricin 01 Following magnetic liposome separation, three eluents were obtained (Fig. 1). All of the fractions were analysed for antimicrobial activity using E. coli CICC 10300 as the indicator strain. Two fractions (2 and 3) exhibited antimicrobial activity. Peak 3, eluting at 18 min, gave the highest antimicrobial activity (Fig. 1A), therefore purification and characterisation were conducted on peak 3 (Fig. 1B). HPLC with the purified sample showed only one peak corresponding to peak 3 (Fig. 2).
3.2. Structural characterisation of andricin 01 Following homogenisation of peak 3, MALDI-TOF/MS analysis yielded a compound with an m/z of 955.1 Da (Fig. 3). The 10-amino acid sequence was successfully
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identified as Ala-Ile-Gly-His-Cys-Leu-Gly-Ala-Thr-Leu by N-terminal sequencing. A similarity search was performed to compare the sequence of amino acids, but no significant similarity was found with previously deposited sequences. Hence, the AMP is safely assigned as a novel peptide and was named andricin 01.
Pertinent andricin 01 physical and chemical attributes were also predicted (Table 2). Based on the sequence, the theoretical isoelectric point was calculated to be 6.78. The GRAVY value is 1.350 and the instability index is 19.77.
The CD data collected were found to be similar to a random coil spectrum with negative bands uncovered near 195 nm and very low ellipticity above 210 nm (Fig. 4). The CD spectra showed similar shapes at different pH values. From this, a three-dimensional rendering was rendered of the secondary structure of andricin 01 (Fig. 5).
3.3. Activity andricin 01 The activity spectrum and activity of andricin 01 (synthetic) are listed in Table 1. All of the bacterial species were found to be sensitive to the AMP. Pseudomonas aeruginosa and Enterobacter cloacae were found to be the most sensitive (MIC = 4 g/mL; MBC = 8 g/mL). However, andricin 01 did not exhibit antimicrobial activity
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against fungi at concentrations up to 100 g/mL (Table 1). Four E. coli strains were evaluated and each showed the same inhibitory activity.
Heat and freeze–thaw treatment did not affect the activity of andricin 01 and it was effective within the range pH 3–9. Moreover, surfactants were not able to decrease the activity of andricin 01 (Table 3).
3.4. Safety evaluation of andricin 01 Cytotoxicity studies on andricin 01 by MTT assay showed that the viability of HL-7702 and HREpiC cells was 86.8% and 82.3%, respectively, in the presence of 100 g/mL andricin 01 (Table 4). The results suggest that andricin 01 may be relatively safe. No haemoglobin release was observed in human red blood cells following incubation at varying peptide concentrations (0–50 g/mL) for 3 h at 37 C, suggesting that andricin 01 will not lyse human red blood cells.
4. Discussion Andrias davidianus is recognised widely in folk medicine as a panacea to multifarious ailments, as far ranging as colds to arthritis, and is even cited as a safeguard against cancer [12,13]. However, until now, AMPs in these ancient balms have not been well characterised. To the best of our knowledge, this is the first attempt to understand the
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scientific properties of the mucus of these animals. Based on this study, there is potential room for future exploration of additional amphibian extractions. For example, there may be value in studying peptides from frogs, such as esculentin-1, esculentin-2, brevinin-1 and brevinin-2 from Odorrana grahami, and nigrocin-2 from Odorrana hosii [6–10].
Recently, efforts have been made to rapidly screen for and identify novel AMPs [1–5,10]. For example, cell membrane chromatography with a silica gel carrier has been used to purify AMPs [30]. Tang et al. modified this method with the lipids from E. coli cell membranes [18]. These methods also have some drawbacks. For example, the combination of silica gel carrier may become unstable [17,19,30] and this kind of method requires complex centrifugation and column loading steps [18,21,22]. In the current study, a magnetic liposome was constructed to screen and purify the AMP. This method is gentle, efficient and convenient.
An interesting characteristic of andricin 01 is that is comprised of only ten amino acids; at <1 kDa mass, its mass is low relative to most proteins. Previous studies of antimicrobials uncovered low mass molecules that lacked complex secondary and tertiary structures [17–21]. For example, studies have uncovered jineol, an alkaloid with cytotoxic activity at 161.05 Da [31], and lacrain from centipedes possessing activity against Gram-negative bacteria [4] at 925.5 Da. Given the shortness of the
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sequences, it is improbable that these peptides can form secondary structures. The difficulty in structure–function similarity searches is compounded by no higher order structures existing [4,17–21,31]. Indeed, similarity searches between andricin 01 and other published molecular sequences were futile. This lends credence that andricin 01 may be a radically different type of AMP all together.
Despite its brevity with a high degree of freedom, andricin 01 appears to be well defined. In bioequivalent media tested, the N-terminus possessed a protonated R side group (His) and a positive charge. However, the charges are thought to be balanced by the non-polar amino acid residues. When the pH value changed, there was little variation in the structure of andricin 01 (Fig. 4); the N-terminal tended to retain a positive charge in the pH range studied, and the carboxy-terminal remained non-protonated, maintaining the negative charge. The charge-induced folding may influence the primary structure. It is believed the overall effect is stabilisation of the zwitterion. Full spectroscopic analysis, i.e. through nuclear magnetic resonance (NMR), has not been conducted and would need to be done for full structural elucidation.
Andricin 01 demonstrated bactericidal activity against Gram-negative bacteria (E. coli, Pseudomonas aeruginosa, Alcaligenes faecalis, Serratia marcescens, E. cloacae and Salmonella Paratyphi B) and Gram-positive bacteria (Bacillus subtilis, Bacillus
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megaterium, Listeria innocua and Staphylococous aureus). It is thought that andricin 01 may interact with the cell envelopes both through hydrophobic and electrostatic interactions. For example, negatively charged phospholipids and teichoic acids on the outer surface of the peptidoglycan in Gram-positive bacteria and negatively charged lipid A of lipopolysaccharide in Gram-negative bacteria may contribute [21–25].
Compared with odorranain-NR (an AMP from amphibian skin), andricin had lower MICs against Gram-negative bacteria; indeed, andricin 01 demonstrated antimicrobial activity against S. marcescens that was not seen in odorranain-NR [32]. Compared with the antimicrobial activity of esculentin-2PLa and esculentin-2 [7,33], andricin 01 was more potent against Gram-negative bacteria. We therefore believe that andricin 01 has real potential as an agent active against Gram-negative bacteria.
From this experimental work, andricin 01 (at 1 MBC) was found to be safe by MTT assay and had no impact on haemolytic activity. Investigation of andricin 01 and similar compounds may pave the new way for novel drug development.
Funding: This study was funded by the Research Foundation of Science and Technology Bureau of Shaanxi, China [2015SZS-15-05], the Research Foundation of Education Bureau of Shaanxi, China [16JS021], the China Scholarship Council (CSC)
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Foundation [201608615024] and the Special Science Foundation of Shaanxi University of Technology [SLGKYQD2014-2-18].
Competing interests: None declared.
Ethical approval: Not required.
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[26] Malheiros PS, Micheletto YMS, Silveira NP, Brandelli A. Development and characterization of phosphatidylcholine nanovesicles containing the antimicrobial peptide nisin. Food Res Int 2010;43:1198–203. [27] Yue TL, Pei JJ, Yuan YH. Purification and characterization of anti-Alicyclobacillus bacteriocin produced by Lactobacillus rhamnosus. J Food Prot 2013;76:1575–81. [28] Kanda H, Kurosaka Y, Fujikawa K, Chiba M, Yamachika S, Okumura R. In vitro and in vivo antibacterial activity of sitafloxacin. Chinese J New Drugs 2013;22:1400–5. [29] Nakashima H, Ooshima T, Kuriyama T. Safety evaluation of inorganic antimicrobial agents (1): analysis of inorganic antimicrobial agents used in antimicrobial product. Biomed Res Trace Elem 2006;17:427–30. [30] Pu C, Tang W. Affinity and selectivity of anchovy antibacterial peptide for Staphylococcus aureus, cell membrane lipid and its application in whole milk. Food Control 2016;72:153–63. [31] Moon SS, Cho N, Jongheon S, Youngwan S, Ock L, Sang, UC. Jineol, a cytotoxic alkaloid from the centipede Scolopendra subspinipes. J Nat Prod 1996;59:777–9. [32] Che Q, Zhou Y, Yang H, Li J, Xu X, Lai R. A novel antimicrobial peptide from amphibian skin secretions of Odorrana grahami. Peptides 2008;29:529–35.
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[33] Sancar-Bas S, Bolkent S. Esculentin-2PLa, a frog skin antimicrobial peptide, causes necrotic cell death in breast cancer cell lines. Eur J Cancer 2016;61:132–3.
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Fig. 1. Separation of antimicrobial peptides from mucus of the Chinese giant salamander (Andrias davidianus) by magnetic nanoliposome separation. (b) Antimicrobial activity against Escherichia coli CICC 10003 of the fractions obtained.
Fig. 2. Reverse-phase high-performance liquid chromatography (RP-HPLC) of peak 3 from the magnetic nanoliposome system.
Fig. 3. Mass spectrum of andricin 01.
Fig. 4. Circular dichroism spectrum of andricin 01.
Fig. 5. Three-dimensional structure of andricin 01.
Table 1 Activity of andricin 01 against various species Micro-organism
MIC (g/mL) [M] MBC (g/mL) [M]
Gram-positive bacteria Bacillus subtilis CICC 10034
32 [33.5]
32 [33.5]
Bacillus megaterium CICC 10448
8 [8.3]
16 [16.7]
Staphylococcus aureus CICC 10306
32 [33.5]
32 [33.5]
S. aureus CICC 10384
32 [33.5]
32 [33.5]
Listeria innocua CICC 10417
32 [33.5]
32 [33.5]
Gram-negative bacteria
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Escherichia coli CICC 10293
8 [8.3]
16 [16.7]
E. coli CGMCC 3373
8 [8.3]
16 [16.7]
E. coli CICC 10302
8 [8.3]
16 [16.7]
E. coli CICC 10300
8 [8.3]
16 [16.7]
Pseudomonas aeruginosa CICC 21636 4 [4.2]
8 [8.3]
Alcaligenes faecalis ATCC 8750
8 [8.3]
16 [16.7]
Serratia marcescens ATCC 4112
8 [8.3]
8 [8.3]
Enterobacter cloacae CICC 21539
4 [4.2]
8 [8.3]
Salmonella Paratyphi CICC 10437
8 [8.3]
16 [16.7]
Aspergillus niger CICC 2124
NA
NA
Candida albicans CICC 1965
NA
NA
Saccharomyces cerevisiae CICC 1002
NA
NA
Fungi
MIC, minimum inhibitory concentration; MBC, minimum bacterial concentration; CICC, China Center of Industrial Culture Collection; CGMCC, China General Microbiological Culture Collection Center; ATCC, American Type Culture Collection; NA, no inhibitory activity.
Table 2 Physical and chemical attributes of andricin 01 Theoretical pI
6.78
GRAVY value
1.350
Estimated half-life 4.4 h reticulocytes in vitro; 20 h yeast in vivo Instability index
19.77
Aliphatic index
137.00
pI, isoelectric point.
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Table 3 Characterisation of andricin 01 Condition
Activity
60 C, 80 C, 100 C
+
Freeze–thaw
+
pH 2, pH 10
–
pH 3–9
+
EDTA, Tween 20 and NaCl
+
EDTA, ethylene-diamine tetra-acetic acid.
Table 4 Safety evaluation of andricin 01 using a human hepatocyte cell line (HL-7702) and human renal epithelial cells (HREpiC) Andricin 01 concentration Viability (%) HL-7702
Haemolysis (%) HREpiC
25 g/mL
100.0 ± 0.30 100.1 ± 0.20 Negative
50 g/mL
100.5 ± 0.52 103.1 ± 1.20 Negative
100 g/mL
86.8 ± 2.10
82.3 ± 1.84
4.3 ± 0.16
200 g/mL
52.1 ± 1.81
50.3 ± 2.05
16.3 ± 2.16
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S0924-8579(17)30138-3 http://dx.doi.org/doi: 10.1016/j.ijantimicag.2017.02.013 ANTAGE 5093
To appear in:
International Journal of Antimicrobial Agents
Received date: Accepted date:
19-7-2016 10-2-2017
Please cite this article as: Jinjin Pei, Lei Jiang, Antimicrobial peptide from mucus of Andrias davidianus: screening and purification by magnetic cell membrane separation technique, International Journal of Antimicrobial Agents (2017), http://dx.doi.org/doi: 10.1016/j.ijantimicag.2017.02.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Antimicrobial peptide from mucus of Andrias davidianus: screening and purification by magnetic cell membrane separation technique
Jinjin Pei a,*, Lei Jiang b
a
Shaanxi Key Laboratory of Biology and Bioresources, Shaanxi University of
Technology, Chaoyang Road, Shaanxi University of Technology, Hanzhong, Shaanxi 723001, China b
Key Laboratory of Tibetan Medicine Research, Northwest Institute of Plateau
Biology, Chinese Academy of Sciences, Xining, Qinghai, China
ARTICLE INFO Article history: Received 19 July 2016 Accepted 10 February 2017
Keywords: Andrias davidianus Mucus Antimicrobial peptide Magnetic cell membrane separation
Page 1 of 25
Escherichia coli
* Corresponding author. Tel.: +86 916 264 1641. E-mail address: [email protected] (J. Pei).
Page 2 of 25
Highlights
An antimicrobial peptide (andricin 01) was isolated from the mucus of Andrias davidianus.
Andricin 01 was purified using an innovative magnetic cell membrane separation technique.
Andricin 01 is a novel antimicrobial peptide.
ABSTRACT Andrias davidianus, the Chinese giant salamander, has been used in traditional Chinese medicine for many decades. However, no antimicrobial peptides (AMPs) have been described from A. davidianus until now. Here we describe a novel AMP (andricin 01) isolated from the mucus of A. davidianus. The peptide was recovered using an innovative magnetic cell membrane separation technique and was characterised using mass spectrometry and circular dichroism (CD) spectroscopy. Andricin 01 is comprised of ten amino acid residues with a total molecular mass of 955.1 Da. CD spectrum analysis gave results similar to the archetypal random coil spectrum, consistent with the three-dimensional rendering calculated by current bioinformatics tools. Andricin 01 was found to be inhibitory both to Gram-negative and Gram-positive bacteria. Furthermore, the peptide at the minimal bacterial concentration did not show cell cytotoxicity against human hepatocytes or renal cells
Page 3 of 25
and did not show haemolytic activity against red blood cells, indicating that is potentially safe and effective for human use. Andricin 01 shows promise as a novel antibacterial that may provide an insight into the development of new drugs.
Page 4 of 25
1. Introduction In recent years, scientists have turned increasingly towards the study of plants and animals in the hope of identifying and characterising new antimicrobial peptides (AMPs) [1–5]. The reason for this is simple. Microbes reproduce and evolve at an exponential rate that is outpacing the discovery of new antimicrobial agents [4]. The emergence of strains of human pathogens that are resistant to most or all of the current lines of treatment [4,5] underpins the increasing need for better treatments [1–5].
Recent research has allowed us to elucidate the primary structure of >8000 AMPs (http://www.bicnirrh.res.in/antimicrobial) [4–10]. Amphibians comprise the oldest class of animals from which these compounds have been isolated [5–10]. Most of the environments that amphibians inhabit are damp and dark, favouring the survival of many pathogenic micro-organisms [6,7]. Accordingly, amphibians have adopted a complex system of AMPs in their immune system to defend against such attackers [8–10].
Andrias davidianus (Chinese giant salamander) is thought to have originated ca. 350 000 000 years ago [11]. Its skin secretes white mucus on stimulation; in regional folk medicine, this by-product is purported to reduce the symptoms and signs of dementia, apoplexy, tetanus and tuberculosis and to improve the outcome of various other
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disease states [12–14]. Until now, there has been little study of the potential bioactive peptides of salamander mucus. The purpose of this study was to evaluate potential antimicrobial components of A. davidianus mucus as potential treatments for human infections.
The methods employed to isolate peptides from heterogeneous mixtures frequently involve complex chromatographic procedures that are labour- and resource-intensive and are subject to failure [15–18]. More rapid and efficient methods to isolate and analyse AMPs are therefore required. Commonality in effective AMPs can and should be exploited [19,20]. For example, it is believed that all AMPs, regardless of their mechanism of action, require an interaction with the targeted bacterial cellular membrane for activity to be initiated [21–24]. Against this background, we aimed to rapidly screen for AMPs that are capable of interfacing with bacterial membranes using immobilised bacterial membrane liposome chromatography. Finally, peptide characteristics including structural parameters, activity spectra, haemolytic capability and cytotoxicity were investigated.
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2. Materials and methods 2.1. Preparation of magnetic nanoliposomes Ferromagnetic Fe3O4 nanoparticles were prepared by a hydrothermal method [25]. Lipid of Escherichia coli was extracted using 2:1 (v/v) chloroform:methanol 18]. Nanomagnetic liposome particles were prepared by the thin-film dispersion method [26].
2.2. Purification of antimicrobial peptides from mucus of Andrias davidianus Lyophilised powder of A. davidianus mucus was dissolved in phosphate-buffered saline (PBS) (pH 7.2, 10 mmol/L) with 50 mmol/L NaCl, was filtered using a cellulose membrane (Ultracel PLGC04310; Merck Millipore, Billerica, MA) with a cut-off molecular weight at 10 kDa and was then dialysed with a 100 Da membrane (SP131048; Yuanye Bio, Shanghai, China) to obtain the crude sample. Magnetic nanoliposomes were then added and were co-cultured at 37 C for 24 h. After collection of magnetic liposomes by a magnet column, PBS (pH 7.2, 10 mM) with 50 mM NaCl served as an isocratic mobile phase at 25 C. Absorbance was recorded at 215 nm using a UV/Vis DAD (Agilent 1260 series; Agilent Technologies, Santa Clara, CA). Peaks were detected and were analysed for their potential bactericidal effect against E. coli CICC 10300 by the agar diffusion method [27]. From this, the fraction that exhibited the highest activity was put through reverse-phase high-performance
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liquid chromatography (RP-HPLC) (Waters Symmetry C18 column, 250 4.6 mm, 5 m; Waters, Dublin, Ireland). A gradient method was used for separation (with mobile phase B, 5−100% in 40 min) at a flow rate of 0.5 mL/min at 30 ± 2 C. The two mobile phases used were: mobile phase (A) 0.05% (v/v) trifluoroacetic acid; and mobile phase (B) 100% acetonitrile.
2.3. Structural characterisation The purified AMP (andricin 01) was tested by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). The sequence was searched for similarity against the NCBI BLAST database (http://blast.ncbi.nlm.nih.gov/) as well as databases accessible through the Bioinformatics Resource Portal ExPASy (http://www.expasy.org). Measurements were taken using circular dichroism (CD) spectroscopy on a Jasco J-810 CD spectrometer (Jasco Co., Tokyo, Japan) with a 0.5 mm path length at UV 195–250 nm at Shaanxi Normal University (Xi'an, China). Molecular stimulation software HyperChem 8.0 (HyperCube Inc., Gainesville, FL) was used to predict the structure under the lowest energy state.
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2.4. Synthesis of peptides AMPs were synthesised by Beijing Huada Protein Engineering Ltd. (Beijing, China) [27]. MALDI-TOF/MS and RP-HPLC were used to confirm peptide purity and identity.
2.5. Activity of the antimicrobial peptide The activity of andricin 01 was tested against the indicator strains listed in Table 1. An increase in optical density at 600 nm was used to monitor growth. Poor nutrient broth and poor potato dextrose broth (0.5 PDB) were used to culture bacteria. The minimum inhibitory concentration (MIC), defined as the lowest concentration of AMP needed to inhibit growth, was observed following incubation at 30 C for 18 h [27], and the minimum bacterial concentration (MBC) was defined as the lowest AMP concentration of AMP with no visual growth observed after 96 h of incubation at 30 C. Tests were conducted three times.
2.6. Safety evaluation The cytotoxicity of andricin 01 was evaluated by the MTT cell viability assay with a human hepatocyte cell line (HL-7702) and human renal epithelial cells (HREpiC) (ATCC, Manassas, VA) [28,29]. Percent haemolysis was determined with respect to a lysis control (red blood cells in 0.1% Triton X-100) and a negative control (in PBS). Assays were conducted in triplicate [18].
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2.7. Statistics MALDI-TOF/MS results were interpreted using Mass Lynx 4.1 software (Waters, Milford, MA) to calculate accurate molecular masses [4]. SPSS Statistics v.18.0 (SPSS Inc., Chicago, IL) was used for data analysis [18]. Data were averaged across replicates. Results are reported as the mean ± standard deviation.
3. Results 3.1. Purification of andricin 01 Following magnetic liposome separation, three eluents were obtained (Fig. 1). All of the fractions were analysed for antimicrobial activity using E. coli CICC 10300 as the indicator strain. Two fractions (2 and 3) exhibited antimicrobial activity. Peak 3, eluting at 18 min, gave the highest antimicrobial activity (Fig. 1A), therefore purification and characterisation were conducted on peak 3 (Fig. 1B). HPLC with the purified sample showed only one peak corresponding to peak 3 (Fig. 2).
3.2. Structural characterisation of andricin 01 Following homogenisation of peak 3, MALDI-TOF/MS analysis yielded a compound with an m/z of 955.1 Da (Fig. 3). The 10-amino acid sequence was successfully
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identified as Ala-Ile-Gly-His-Cys-Leu-Gly-Ala-Thr-Leu by N-terminal sequencing. A similarity search was performed to compare the sequence of amino acids, but no significant similarity was found with previously deposited sequences. Hence, the AMP is safely assigned as a novel peptide and was named andricin 01.
Pertinent andricin 01 physical and chemical attributes were also predicted (Table 2). Based on the sequence, the theoretical isoelectric point was calculated to be 6.78. The GRAVY value is 1.350 and the instability index is 19.77.
The CD data collected were found to be similar to a random coil spectrum with negative bands uncovered near 195 nm and very low ellipticity above 210 nm (Fig. 4). The CD spectra showed similar shapes at different pH values. From this, a three-dimensional rendering was rendered of the secondary structure of andricin 01 (Fig. 5).
3.3. Activity andricin 01 The activity spectrum and activity of andricin 01 (synthetic) are listed in Table 1. All of the bacterial species were found to be sensitive to the AMP. Pseudomonas aeruginosa and Enterobacter cloacae were found to be the most sensitive (MIC = 4 g/mL; MBC = 8 g/mL). However, andricin 01 did not exhibit antimicrobial activity
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against fungi at concentrations up to 100 g/mL (Table 1). Four E. coli strains were evaluated and each showed the same inhibitory activity.
Heat and freeze–thaw treatment did not affect the activity of andricin 01 and it was effective within the range pH 3–9. Moreover, surfactants were not able to decrease the activity of andricin 01 (Table 3).
3.4. Safety evaluation of andricin 01 Cytotoxicity studies on andricin 01 by MTT assay showed that the viability of HL-7702 and HREpiC cells was 86.8% and 82.3%, respectively, in the presence of 100 g/mL andricin 01 (Table 4). The results suggest that andricin 01 may be relatively safe. No haemoglobin release was observed in human red blood cells following incubation at varying peptide concentrations (0–50 g/mL) for 3 h at 37 C, suggesting that andricin 01 will not lyse human red blood cells.
4. Discussion Andrias davidianus is recognised widely in folk medicine as a panacea to multifarious ailments, as far ranging as colds to arthritis, and is even cited as a safeguard against cancer [12,13]. However, until now, AMPs in these ancient balms have not been well characterised. To the best of our knowledge, this is the first attempt to understand the
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scientific properties of the mucus of these animals. Based on this study, there is potential room for future exploration of additional amphibian extractions. For example, there may be value in studying peptides from frogs, such as esculentin-1, esculentin-2, brevinin-1 and brevinin-2 from Odorrana grahami, and nigrocin-2 from Odorrana hosii [6–10].
Recently, efforts have been made to rapidly screen for and identify novel AMPs [1–5,10]. For example, cell membrane chromatography with a silica gel carrier has been used to purify AMPs [30]. Tang et al. modified this method with the lipids from E. coli cell membranes [18]. These methods also have some drawbacks. For example, the combination of silica gel carrier may become unstable [17,19,30] and this kind of method requires complex centrifugation and column loading steps [18,21,22]. In the current study, a magnetic liposome was constructed to screen and purify the AMP. This method is gentle, efficient and convenient.
An interesting characteristic of andricin 01 is that is comprised of only ten amino acids; at <1 kDa mass, its mass is low relative to most proteins. Previous studies of antimicrobials uncovered low mass molecules that lacked complex secondary and tertiary structures [17–21]. For example, studies have uncovered jineol, an alkaloid with cytotoxic activity at 161.05 Da [31], and lacrain from centipedes possessing activity against Gram-negative bacteria [4] at 925.5 Da. Given the shortness of the
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sequences, it is improbable that these peptides can form secondary structures. The difficulty in structure–function similarity searches is compounded by no higher order structures existing [4,17–21,31]. Indeed, similarity searches between andricin 01 and other published molecular sequences were futile. This lends credence that andricin 01 may be a radically different type of AMP all together.
Despite its brevity with a high degree of freedom, andricin 01 appears to be well defined. In bioequivalent media tested, the N-terminus possessed a protonated R side group (His) and a positive charge. However, the charges are thought to be balanced by the non-polar amino acid residues. When the pH value changed, there was little variation in the structure of andricin 01 (Fig. 4); the N-terminal tended to retain a positive charge in the pH range studied, and the carboxy-terminal remained non-protonated, maintaining the negative charge. The charge-induced folding may influence the primary structure. It is believed the overall effect is stabilisation of the zwitterion. Full spectroscopic analysis, i.e. through nuclear magnetic resonance (NMR), has not been conducted and would need to be done for full structural elucidation.
Andricin 01 demonstrated bactericidal activity against Gram-negative bacteria (E. coli, Pseudomonas aeruginosa, Alcaligenes faecalis, Serratia marcescens, E. cloacae and Salmonella Paratyphi B) and Gram-positive bacteria (Bacillus subtilis, Bacillus
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megaterium, Listeria innocua and Staphylococous aureus). It is thought that andricin 01 may interact with the cell envelopes both through hydrophobic and electrostatic interactions. For example, negatively charged phospholipids and teichoic acids on the outer surface of the peptidoglycan in Gram-positive bacteria and negatively charged lipid A of lipopolysaccharide in Gram-negative bacteria may contribute [21–25].
Compared with odorranain-NR (an AMP from amphibian skin), andricin had lower MICs against Gram-negative bacteria; indeed, andricin 01 demonstrated antimicrobial activity against S. marcescens that was not seen in odorranain-NR [32]. Compared with the antimicrobial activity of esculentin-2PLa and esculentin-2 [7,33], andricin 01 was more potent against Gram-negative bacteria. We therefore believe that andricin 01 has real potential as an agent active against Gram-negative bacteria.
From this experimental work, andricin 01 (at 1 MBC) was found to be safe by MTT assay and had no impact on haemolytic activity. Investigation of andricin 01 and similar compounds may pave the new way for novel drug development.
Funding: This study was funded by the Research Foundation of Science and Technology Bureau of Shaanxi, China [2015SZS-15-05], the Research Foundation of Education Bureau of Shaanxi, China [16JS021], the China Scholarship Council (CSC)
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Foundation [201608615024] and the Special Science Foundation of Shaanxi University of Technology [SLGKYQD2014-2-18].
Competing interests: None declared.
Ethical approval: Not required.
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[26] Malheiros PS, Micheletto YMS, Silveira NP, Brandelli A. Development and characterization of phosphatidylcholine nanovesicles containing the antimicrobial peptide nisin. Food Res Int 2010;43:1198–203. [27] Yue TL, Pei JJ, Yuan YH. Purification and characterization of anti-Alicyclobacillus bacteriocin produced by Lactobacillus rhamnosus. J Food Prot 2013;76:1575–81. [28] Kanda H, Kurosaka Y, Fujikawa K, Chiba M, Yamachika S, Okumura R. In vitro and in vivo antibacterial activity of sitafloxacin. Chinese J New Drugs 2013;22:1400–5. [29] Nakashima H, Ooshima T, Kuriyama T. Safety evaluation of inorganic antimicrobial agents (1): analysis of inorganic antimicrobial agents used in antimicrobial product. Biomed Res Trace Elem 2006;17:427–30. [30] Pu C, Tang W. Affinity and selectivity of anchovy antibacterial peptide for Staphylococcus aureus, cell membrane lipid and its application in whole milk. Food Control 2016;72:153–63. [31] Moon SS, Cho N, Jongheon S, Youngwan S, Ock L, Sang, UC. Jineol, a cytotoxic alkaloid from the centipede Scolopendra subspinipes. J Nat Prod 1996;59:777–9. [32] Che Q, Zhou Y, Yang H, Li J, Xu X, Lai R. A novel antimicrobial peptide from amphibian skin secretions of Odorrana grahami. Peptides 2008;29:529–35.
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[33] Sancar-Bas S, Bolkent S. Esculentin-2PLa, a frog skin antimicrobial peptide, causes necrotic cell death in breast cancer cell lines. Eur J Cancer 2016;61:132–3.
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Fig. 1. Separation of antimicrobial peptides from mucus of the Chinese giant salamander (Andrias davidianus) by magnetic nanoliposome separation. (b) Antimicrobial activity against Escherichia coli CICC 10003 of the fractions obtained.
Fig. 2. Reverse-phase high-performance liquid chromatography (RP-HPLC) of peak 3 from the magnetic nanoliposome system.
Fig. 3. Mass spectrum of andricin 01.
Fig. 4. Circular dichroism spectrum of andricin 01.
Fig. 5. Three-dimensional structure of andricin 01.
Table 1 Activity of andricin 01 against various species Micro-organism
MIC (g/mL) [M] MBC (g/mL) [M]
Gram-positive bacteria Bacillus subtilis CICC 10034
32 [33.5]
32 [33.5]
Bacillus megaterium CICC 10448
8 [8.3]
16 [16.7]
Staphylococcus aureus CICC 10306
32 [33.5]
32 [33.5]
S. aureus CICC 10384
32 [33.5]
32 [33.5]
Listeria innocua CICC 10417
32 [33.5]
32 [33.5]
Gram-negative bacteria
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Escherichia coli CICC 10293
8 [8.3]
16 [16.7]
E. coli CGMCC 3373
8 [8.3]
16 [16.7]
E. coli CICC 10302
8 [8.3]
16 [16.7]
E. coli CICC 10300
8 [8.3]
16 [16.7]
Pseudomonas aeruginosa CICC 21636 4 [4.2]
8 [8.3]
Alcaligenes faecalis ATCC 8750
8 [8.3]
16 [16.7]
Serratia marcescens ATCC 4112
8 [8.3]
8 [8.3]
Enterobacter cloacae CICC 21539
4 [4.2]
8 [8.3]
Salmonella Paratyphi CICC 10437
8 [8.3]
16 [16.7]
Aspergillus niger CICC 2124
NA
NA
Candida albicans CICC 1965
NA
NA
Saccharomyces cerevisiae CICC 1002
NA
NA
Fungi
MIC, minimum inhibitory concentration; MBC, minimum bacterial concentration; CICC, China Center of Industrial Culture Collection; CGMCC, China General Microbiological Culture Collection Center; ATCC, American Type Culture Collection; NA, no inhibitory activity.
Table 2 Physical and chemical attributes of andricin 01 Theoretical pI
6.78
GRAVY value
1.350
Estimated half-life 4.4 h reticulocytes in vitro; 20 h yeast in vivo Instability index
19.77
Aliphatic index
137.00
pI, isoelectric point.
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Table 3 Characterisation of andricin 01 Condition
Activity
60 C, 80 C, 100 C
+
Freeze–thaw
+
pH 2, pH 10
–
pH 3–9
+
EDTA, Tween 20 and NaCl
+
EDTA, ethylene-diamine tetra-acetic acid.
Table 4 Safety evaluation of andricin 01 using a human hepatocyte cell line (HL-7702) and human renal epithelial cells (HREpiC) Andricin 01 concentration Viability (%) HL-7702
Haemolysis (%) HREpiC
25 g/mL
100.0 ± 0.30 100.1 ± 0.20 Negative
50 g/mL
100.5 ± 0.52 103.1 ± 1.20 Negative
100 g/mL
86.8 ± 2.10
82.3 ± 1.84
4.3 ± 0.16
200 g/mL
52.1 ± 1.81
50.3 ± 2.05
16.3 ± 2.16
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