Reduction of Oxidative Stress through Activating the Nrf2 mediated HO-1 Antioxidant Efficacy Signaling Pathway by MS15, an Antimicrobial Peptide from Bacillus velezensis
Abstract
:1. Introduction
2. Materials and Methods
2.1. Reagents and Materials
2.2. In Vitro Process of Isolation, Screening, and Culture Media Optimization
2.3. Optimizations of Culture Media Design by Response Surface Methodology and Statistical Analysis
2.4. Culture and Purification of Peptide MS15
2.5. Electrophoresis with Tricine SDS-PAGE and Bioassay (In Situ) Analysis
2.6. MALDI-TOF/MS for Molecular Weight Determination
2.7. pH and Thermal Stability of MS15
2.8. Evaluation of Antimicrobial Susceptibility
2.9. Bacterial Killing Kinetics Assay of Peptide MS15
2.10. DPPH (2,2-Diphenyl-1-Picrylhydrazyl) Radical Scavenging Capability Assay
2.11. ABTS (2,2′-Azino-bis (3-Ethylbenzthiazoline-6-Sulfonic Acid)) Radical Scavenging Capability Assay
2.12. Superoxide Radical Scavenging Capability Assay
2.13. FRAP (Ferric Reducing Antioxidant Power) Activity Assay
2.14. CUPRAC (Cupric Reducing Antioxidant Capacity) Activity Assay
2.15. ORAC (Oxygen Radicle Absorbance Capacity) Assay
2.16. Cellular NO (Nitric Oxide) Measurement in RAW 264.7 Cells
2.17. Intracellular ROS (Reactive Oxygen Species) Measurement in RAW 246.7 Cells
2.18. Cell Culture and Cell Cytotoxicity Assay
2.19. Western Blot Analysis with Cell Lysates
2.20. RT-PCR (Reverse Transcription-Polymerase Chain Reaction) Assay
2.21. Statistical Analysis
3. Results
3.1. Strain Isolation and Identification
3.2. Experimental Design and Box-Behnken Analysis by Response Surface Methodology
3.3. Culture Media, Purification, and Molecular Weight Resolve of MS15
3.4. Mass Spectroscopy Analysis by MALDI-TOF-MS
3.5. Stability Analysis of MS15
3.6. Assessment of Antimicrobial Susceptibility and Time-Kill Kinetics Analyses
3.7. Radical Scavenging Activity Assay
3.8. Reducing Power Measurement Assay
3.9. Cell Viability Assay
3.10. ROS Measurement and Nitric Oxide Inhibitory Assay
3.11. Effects of MS15 on Antioxidant Enzyme in RAW 264.7 Cells
4. Discussion
5. Conclusions
Author Contributions
Funding
Conflicts of Interest
References
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Code | Variables of Choice (Independent) | Up Level | Midpoint | Down Level | Generated Optimum Factor |
---|---|---|---|---|---|
(+1) | (0) | (−1) | |||
A | Soluble starch (g/L) | 25 | 15 | 5 | 13.42 |
B | Yeast extract (g/L) | 15 | 10 | 5 | 7.64 |
C | NaCl (g/L) | 0.15 | 0.1 | 0.05 | 0.089 |
Run | Soluble Starch (g/L) | Yeast Extract (g/L) | NaCl (g/L) | Bacteriocin Activity of MS15 (AU/mL) | |
---|---|---|---|---|---|
A | B | C | Actual Value x | Predicted Value | |
1 | 15 | 10 | 0.1 | 12,138.00 | 12,205.80 |
2 | 5 | 15 | 0.1 | 9460.00 | 9166.00 |
3 | 15 | 10 | 0.1 | 11,940.00 | 12,205.80 |
4 | 5 | 10 | 0.05 | 8650.00 | 8782.25 |
5 | 15 | 5 | 0.05 | 10,450.00 | 10,072.25 |
6 | 5 | 10 | 0.15 | 10,113.00 | 10,029.25 |
7 | 25 | 10 | 0.05 | 11,146.00 | 11,229.75 |
8 | 15 | 10 | 0.1 | 12,268.00 | 12,205.80 |
9 | 15 | 5 | 0.15 | 11,329.00 | 11,167.25 |
10 | 25 | 5 | 0.1 | 9336.00 | 9630.00 |
11 | 5 | 5 | 0.1 | 7622.00 | 7867.50 |
12 | 15 | 10 | 0.1 | 12,316.00 | 12,205.80 |
13 | 25 | 15 | 0.1 | 11,560.00 | 11,314.50 |
14 | 15 | 15 | 0.05 | 11,742.00 | 11,903.75 |
15 | 15 | 15 | 0.15 | 11,941.00 | 12,318.75 |
16 | 25 | 10 | 0.15 | 11,625.00 | 11,492.75 |
17 | 15 | 10 | 0.1 | 12,367.00 | 12,205.80 |
Purification Steps | Vol (mL) | Total Protein (mg) | Total Activity (AU) | Specific Activity (AU/mg) | Purification Fold | Recovery (%) |
---|---|---|---|---|---|---|
Cell free crude sample | 935 | 388.5 | 270,000 | 694.98 | 1 | 100 |
Ammonium sulphate pallet aliquots | 52 | 98.45 | 156,000 | 1584.56 | 2.28 | 57.78 |
Sephadex G-50 gel | 8 | 7.23 | 38,000 | 5255.88 | 7.56 | 14.07 |
DEAE Sephadex A-50 gel | 2 | 0.94 | 16,000 | 17,021.27 | 24.49 | 5.92 |
Microorganisms | MIC of MS15 (µg/mL) | MBC of MS15 (µg/mL) | ||
---|---|---|---|---|
MS15 | Bacitracin | Vancomycin | ||
Gram-negative bacteria | ||||
Escherichia coli KCTC 1923 | 40 | 160 | 80 | 80 |
Pseudomonas aeruginosa KCTC 1637 | 160 | >160 | >160 | 320 |
Salmonella typhimurium KCTC 1925 | 40 | 80 | 40 | 80 |
Alcaligenes faecalis ATCC 1004 | 160 | 160 | 80 | 320 |
Extended-spectrum beta-lactamase V4 (Escherichia coli) | 80 | 160 | 80 | 80 |
Extended-spectrum beta-lactamase W1 | 40 | 80 | 40 | 80 |
Extended-spectrum beta-lactamase 31 | 40 | 80 | 40 | 80 |
Gram-positive bacteria | ||||
Vancomycin-resistant Enterococci 4 | 160 | 80 | 160 | 320 |
Vancomycin-resistant Enterococci 89 | 80 | >160 | >160 | 160 |
Vancomycin-resistant Enterococci 98 | 80 | >160 | >160 | 160 |
Staphylococcus aureus KCTC 1928 | 160 | >160 | >160 | 160 |
Methicillin-resistant Staphylococcus aureus 5-3 | 2.5 | 5 | 2.5 | 5 |
Methicillin-resistant Staphylococcus aureus B15 | 40 | 160 | 80 | 120 |
Mycobacterium smegmatis ATCC 9341 | 20 | 40 | 2.5 | 40 |
Micrococcus luteus ATCC 9341 | 40 | 40 | 2.5 | 40 |
Enterococcus faecalis ATCC 29212 | 20 | 5 | 2.5 | 20 |
Bacillus subtilis ATCC 6633 | 10 | 20 | 0.8 | 20 |
Vancomycin-resistant Staphylococcus aureus | 80 | 160 | >160 | 160 |
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Khan, M.M.; Kim, Y.K.; Bilkis, T.; Suh, J.-W.; Lee, D.Y.; Yoo, J.C. Reduction of Oxidative Stress through Activating the Nrf2 mediated HO-1 Antioxidant Efficacy Signaling Pathway by MS15, an Antimicrobial Peptide from Bacillus velezensis. Antioxidants 2020, 9, 934. https://doi.org/10.3390/antiox9100934
Khan MM, Kim YK, Bilkis T, Suh J-W, Lee DY, Yoo JC. Reduction of Oxidative Stress through Activating the Nrf2 mediated HO-1 Antioxidant Efficacy Signaling Pathway by MS15, an Antimicrobial Peptide from Bacillus velezensis. Antioxidants. 2020; 9(10):934. https://doi.org/10.3390/antiox9100934
Chicago/Turabian StyleKhan, Md Maruf, Young Kyun Kim, Tahmina Bilkis, Joo-Won Suh, Dae Young Lee, and Jin Cheol Yoo. 2020. "Reduction of Oxidative Stress through Activating the Nrf2 mediated HO-1 Antioxidant Efficacy Signaling Pathway by MS15, an Antimicrobial Peptide from Bacillus velezensis" Antioxidants 9, no. 10: 934. https://doi.org/10.3390/antiox9100934