Enhanced Antibacterial, Anti-Inflammatory, and Antibiofilm Activities of Tryptophan-Substituted Peptides Derived from Cecropin A-Melittin Hybrid Peptide BP100
Abstract
:1. Introduction
2. Results and Discussion
2.1. Peptide Design and Characterization
2.2. Antibacterial Activity and Cell Selectivity
2.3. Anti-Inflammatory Activity
2.4. Antibiofilm Activity
2.5. Salt and Serum Stability
2.6. Secondary Structures of BP100 and Selected Analogs
2.7. Antibacterial Mechanism Studies
2.7.1. Membrane Depolarization
2.7.2. Outer Membrane Permeability
2.7.3. Inner Membrane Permeabilization
2.8. Synergistic Antibacterial Effect with Ciprofloxacin
3. Materials and Methods
3.1. Materials
3.2. Bacterial Strains
3.3. Peptide Synthesis
3.4. Antimicrobial Activity Assay
3.5. Hemolytic Activity Assay
3.6. Cytotoxicity Assay
3.7. Measurement of TNF-α and IL-6 Release from LPS-Stimulated RAW264.7 Cells
3.8. Biofilm Inhibition Assay (MBIC)
3.9. Biofilm Eradication Assay (MBEC)
3.10. Circular Dichroism (CD)
3.11. Outer Membrane Permeability
3.12. Cytoplasmic Membrane Depolarization Assay
3.13. Flow Cytometry Analysis
3.14. Synergy Testing using Checkerboard Assay
3.15. Statistical Analysis
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Peptides | Amino Acid Sequences a | Molecular Mass (Da) | Net Charge | μH b | |
---|---|---|---|---|---|
Calculated | Observed | ||||
BP100 | KKLFKKILKYL-NH2 | 1421.87 | 1420.74 | +5 | 0.417 |
BP1 | KKLWKKILKYL-NH2 | 1460.91 | 1459.91 | +5 | 0.469 |
BP2 | KKLFKKWLKYL-NH2 | 1494.93 | 1493.80 | +5 | 0.468 |
BP3 | KKLFKKIWKYL-NH2 | 1494.93 | 1493.77 | +5 | 0.477 |
BP4 | KKLFKKILKWL-NH2 | 1444.91 | 1443.87 | +5 | 0.545 |
BP5 * | KKWWKKILKYL-NH2 | 1533.96 | 1532.86 | +5 | 0.519 |
BP6 * | KKWFKKILKWL-NH2 | 1517.96 | 1516.80 | +5 | 0.595 |
BP7 | KKLWKKIWKYL-NH2 | 1533.96 | 1532.98 | +5 | 0.519 |
BP8 * | KKLWKKILKWL-NH2 | 1483.95 | 1482.94 | +5 | 0.586 |
BP9 | KKLFKKWWKYL-NH2 | 1567.98 | 1566.80 | +5 | 0.518 |
BP10 | KKLFKKWLKYW-NH2 | 1567.98 | 1566.78 | +5 | 0.518 |
BP11 * | KKLFKKIWKWL-NH2 | 1517.96 | 1516.99 | +5 | 0.595 |
BP12 | KKLFKKILKWW-NH2 | 1517.96 | 1516.96 | +5 | 0.595 |
BP13 * | KKWFKKWLKWL-NH2 | 1591.02 | 1589.89 | +5 | 0.635 |
BP14 | KKLWKKWLKYW-NH2 | 1607.02 | 1605.80 | +5 | 0.560 |
BP15 | KKLWKKIWKYW-NH2 | 1607.02 | 1605.82 | +5 | 0.569 |
BP16 | KKLFKKWWKWL-NH2 | 1591.02 | 1589.69 | +5 | 0.635 |
Bacterial Strains | Minimal Inhibitory Concentration (MIC) a (μM) | |||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
BP100 | BP1 | BP2 | BP3 | BP4 | BP5 | BP6 | BP7 | BP8 | BP9 | BP10 | BP11 | BP12 | BP13 | BP14 | BP15 | BP16 | ME | |
Gram-positive bacteria | ||||||||||||||||||
S. aureus (KCTC 1621) | 4 | 8 | 4 | 8 | 4 | 2 | 2 | 2 | 4 | 8 | 8 | 4 | 4 | 4 | 2 | 2 | 16 | 8 |
S. epidermidis (KCTC 1917) | 4 | 8 | 2 | 8 | 8 | 1 | 2 | 2 | 2 | 4 | 8 | 4 | 4 | 8 | 2 | 4 | 16 | 32 |
B. subtilis (KCTC 3068) | 8 | 8 | 8 | 16 | 4 | 2 | 4 | 4 | 8 | 16 | 16 | 8 | 8 | 8 | 4 | 4 | 16 | 16 |
Resistant Gram-positive bacteria | ||||||||||||||||||
MRSA b (CCARM 3089) | 32 | 32 | 16 | 64 | 64 | 4 | 8 | 8 | 16 | 64 | 64 | 8 | 64 | 32 | 16 | 16 | 16 | 32 |
MRSA (CCARM 3090) | 64 | 64 | 16 | 64 | 64 | 8 | 16 | 32 | 16 | 64 | 64 | 32 | 64 | 32 | 32 | 64 | 128 | 8 |
VREF c (ATCC 51559) | 64 | 64 | 128 | 64 | 16 | 8 | 8 | 32 | 16 | 64 | 128 | 16 | 128 | 16 | 64 | 64 | 64 | 64 |
Gram-negative bacteria | ||||||||||||||||||
E. coli (KCTC 1682) | 4 | 8 | 2 | 8 | 4 | 2 | 4 | 4 | 2 | 4 | 8 | 8 | 8 | 8 | 4 | 4 | 16 | 8 |
P. aeruginosa (KCTC 1637) | 16 | 16 | 8 | 32 | 16 | 4 | 16 | 8 | 16 | 16 | 8 | 8 | 16 | 16 | 8 | 8 | 8 | 16 |
S. typhimurium (KCTC 1926) | 4 | 8 | 8 | 4 | 2 | 2 | 4 | 2 | 4 | 4 | 8 | 2 | 4 | 8 | 8 | 4 | 16 | 16 |
Resistant Gram-negative bacteria | ||||||||||||||||||
MDRPA d (CCARM 2095) | 32 | 16 | 8 | 16 | 8 | 8 | 8 | 8 | 8 | 16 | 16 | 8 | 16 | 8 | 16 | 16 | 32 | 32 |
MDRPA (CCARM 2109) | 16 | 16 | 4 | 16 | 16 | 4 | 16 | 4 | 16 | 16 | 16 | 16 | 16 | 16 | 8 | 8 | 32 | 32 |
GM e | 22.5 | 22.5 | 18.5 | 27.3 | 18.7 | 4.1 | 8.0 | 9.6 | 9.8 | 25.1 | 31.3 | 10.4 | 30.2 | 14.2 | 14.9 | 17.6 | 32.7 | 24.0 |
HC10 f | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | 2.0 |
TI g | 22.7 | 22.7 | 27.7 | 18.8 | 27.4 | 124.9 | 64.0 | 53.3 | 52.2 | 20.4 | 16.4 | 49.2 | 17.0 | 36.1 | 34.4 | 29.1 | 15.7 | 0.08 |
Peptides | MBIC50 (μM)/MBIC90 (μM) | MBEC50 (μM)/MBEC90 (μM) |
---|---|---|
BP100 | 4–8/8 | 16–32/32 |
BP1 | 4–8/8 | 8/16 |
BP2 | 8–16/16 | 16–32/32 |
BP3 | 8–16/16 | 16/32 |
BP4 | 4–8/8 | 8/32 |
BP5 | 4–8/8 | 2/8 |
BP6 | 4–8/8 | 4/8 |
BP7 | 4–8/8 | 4–8/8 |
BP8 | 4–8/8 | 2/8 |
BP9 | 8–16/16 | 4/8 |
BP10 | 16–32/32 | 8–16/16 |
BP11 | 4–8/8 | 4–8/8 |
BP12 | 4–8/8 | 8–16/16 |
BP13 | 4–8/8 | 4/8 |
BP14 | 8–16/16 | 8–16/16 |
BP15 | 4–8/8 | 8–16/16 |
BP16 | 8–16/16 | 16/32 |
LL-37 | 32–64/64 | 32/>64 |
Peptides | Control | 150 mM of NaCl | 4.5 mM of KCl | 6 μM of NH4Cl | 1 mM of MgCl2 | 2.5 mM of CaCl2 | 4 μM of FeCl3 | 10% Human Serum |
---|---|---|---|---|---|---|---|---|
E. coli (KCTC 1682) | ||||||||
BP100 | 4 | 16 | 16 | 16 | 16 | 16 | 16 | 16 |
BP5 | 2 | 8 | 4 | 4 | 4 | 8 | 8 | 4 |
BP6 | 4 | 8 | 4 | 16 | 8 | 16 | 16 | 4 |
BP8 | 2 | 8 | 4 | 8 | 8 | 16 | 4 | 4 |
BP11 | 8 | 16 | 8 | 4 | 8 | 16 | 4 | 8 |
BP13 | 8 | 16 | 8 | 8 | 8 | 16 | 8 | 16 |
S. aureus (KCTC 1621) | ||||||||
BP100 | 4 | 16 | 16 | 16 | 16 | 16 | 16 | 32 |
BP5 | 2 | 4 | 4 | 4 | 4 | 4 | 4 | 4 |
BP6 | 2 | 4 | 4 | 4 | 4 | 4 | 8 | 4 |
BP8 | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 4 |
BP11 | 4 | 8 | 4 | 4 | 4 | 16 | 4 | 8 |
BP13 | 4 | 8 | 8 | 8 | 8 | 8 | 8 | 8 |
Peptides | MICA | [A] | FICA | MICB | [B] | FICB | FICIa | Interpretation |
---|---|---|---|---|---|---|---|---|
BP100 | 32 | 4 | 0.125 | 2048 | 512 | 0.25 | 0.375 | synergy |
BP5 | 8 | 0.5 | 0.0625 | 2048 | 512 | 0.25 | 0.3125 | synergy |
BP6 | 8 | 1 | 0.125 | 2048 | 512 | 0.25 | 0.375 | synergy |
BP8 | 8 | 0.5 | 0.0625 | 2048 | 512 | 0.25 | 0.375 | synergy |
BP11 | 8 | 0.5 | 0.0625 | 2048 | 512 | 0.25 | 0.3125 | synergy |
BP13 | 8 | 0.125 | 0.015625 | 2048 | 512 | 0.25 | 0.2656 | synergy |
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Kumar, S.D.; Kim, E.Y.; Radhakrishnan, N.K.; Bang, J.K.; Yang, S.; Shin, S.Y. Enhanced Antibacterial, Anti-Inflammatory, and Antibiofilm Activities of Tryptophan-Substituted Peptides Derived from Cecropin A-Melittin Hybrid Peptide BP100. Molecules 2024, 29, 5231. https://doi.org/10.3390/molecules29225231
Kumar SD, Kim EY, Radhakrishnan NK, Bang JK, Yang S, Shin SY. Enhanced Antibacterial, Anti-Inflammatory, and Antibiofilm Activities of Tryptophan-Substituted Peptides Derived from Cecropin A-Melittin Hybrid Peptide BP100. Molecules. 2024; 29(22):5231. https://doi.org/10.3390/molecules29225231
Chicago/Turabian StyleKumar, Sukumar Dinesh, Eun Young Kim, Naveen Kumar Radhakrishnan, Jeong Kyu Bang, Sungtae Yang, and Song Yub Shin. 2024. "Enhanced Antibacterial, Anti-Inflammatory, and Antibiofilm Activities of Tryptophan-Substituted Peptides Derived from Cecropin A-Melittin Hybrid Peptide BP100" Molecules 29, no. 22: 5231. https://doi.org/10.3390/molecules29225231
APA StyleKumar, S. D., Kim, E. Y., Radhakrishnan, N. K., Bang, J. K., Yang, S., & Shin, S. Y. (2024). Enhanced Antibacterial, Anti-Inflammatory, and Antibiofilm Activities of Tryptophan-Substituted Peptides Derived from Cecropin A-Melittin Hybrid Peptide BP100. Molecules, 29(22), 5231. https://doi.org/10.3390/molecules29225231