Bioactivity and Bactericidal Mechanism of Histidine-Rich β-Hairpin Peptide Against Gram-Negative Bacteria
Abstract
:1. Introduction
2. Results
2.1. Design and Characterization of the Peptides
2.2. CD Analysis
2.3. Hemolytic Activity
2.4. Cytotoxicity
2.5. Antimicrobial Activity
2.6. Salt Sensitivity
2.7. Antibacterial Mechanism Study
2.7.1. LPS Binding Assay
2.7.2. Outer Membrane Permeability Assay
2.7.3. Inner Membrane Permeability
2.7.4. Membrane Depolarization
2.7.5. Swimming Motility Analysis
2.7.6. SEM
2.7.7. Inhibition of Endotoxin-Induced Inflammation
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Peptide Synthesis and Sequence Analysis
4.3. Circular Dichroism (CD) Measurements
4.4. Measurement of Hemolytic Activity
4.5. Measurement of Cytotoxicity
4.6. Minimum Inhibitory Concentration (MIC) Measurements
4.7. Salt Sensitivity Assays
4.8. Antimicrobial Mechanism Assays
4.8.1. LPS Binding Assay
4.8.2. Outer Membrane Permeability Assay
4.8.3. Inner Membrane Permeability Assay
4.8.4. Membrane Depolarization
4.8.5. Swimming Motility Assay
4.8.6. SEM Characterization
4.8.7. Inhibition of Endotoxin-Induced Inflammation
4.9. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Acknowledgments
Conflicts of Interest
References
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Peptides | Sequence | Theoretical MWa | Measured MW | Retention Time (min) | Hb | μHrelc | Net Charge |
---|---|---|---|---|---|---|---|
HI2 | RRIHIHIDPGIHIHIRR-NH2 | 2023.46 | 2024.47 | 11.13 | −0.35 | 2.39 | +9 |
HW2 | RRWHWHWDPGWHWHWRR-NH2 | 2461.78 | 2461.82 | 10.55 | 0.02 | 2.35 | +9 |
HV2 | RRVHVHVDPGVHVHVRR-NH2 | 1939.29 | 1939.33 | 12.20 | −2.07 | 1.89 | +9 |
HF2 | RRFHFHFDPGFHFHFRR-NH2 | 2227.56 | 2227.59 | 18.06 | 0.13 | 2.53 | +9 |
PG-1 | RGGRLCYCRRFCVCVGR-NH2 | 2155.61 | 2156.35 | 12.56 | −2.42 | 1.37 | +7 |
HI2 | HW2 | HV2 | HF2 | PG-1 | |
---|---|---|---|---|---|
MICa (μM) | |||||
Gram- | |||||
E. coli 25922 | 32 | 4 | 8 | 4 | 4 |
E. coli K88 | 32 | 4 | 4 | 2 | 2 |
E. coli K99 | >128 | 4 | 8 | 4 | 2 |
E. coli UB1005 | 16 | 2 | 8 | 8 | 2 |
S.pullorum C7913 | 16 | 2 | 8 | 4 | 4 |
P. auruginosa 27853 | >128 | 8 | 8 | >128 | 8 |
Gram+ | |||||
S.aureus 29213 | >128 | 4 | >128 | 64 | 2 |
S.aureus 43300 | >128 | 2 | >128 | 4 | 8 |
S.epidermidis 12228 | >128 | 2 | >128 | 4 | 8 |
MBCb (GM) | |||||
Gram (−) | 50.80 | 3.56 | 7.13 | 8.00 | 3.17 |
Gram (+) | 256 | 2.52 | 256 | 10.08 | 5.04 |
Gram (+,−) | 87.09 | 3.17 | 23.52 | 8.64 | 3.70 |
MHCc(µM) | >128 | 8 | >128 | 16 | 4 |
TId | |||||
TI(−) | 5.04 | 2.25 | 35.90 | 2.00 | 1.26 |
TI(+) | 1 | 3.17 | 1 | 1.59 | 0.79 |
TI(+,−) | 2.94 | 2.52 | 10.88 | 1.85 | 1.08 |
Peptides | Control b | NaCl b | KCl b | NH4Cl b | MgCl2 b | ZnCl2 b | FeCl3 b |
---|---|---|---|---|---|---|---|
Gram-negative strain E. coli ATCC25922 | |||||||
HV2 | 8 | 4 | 4 | 4 | 8 | 1 | 4 |
PG-1 | 4 | 4 | 4 | 4 | 4 | 2 | 4 |
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Dong, N.; Wang, C.; Zhang, T.; Zhang, L.; Xue, C.; Feng, X.; Bi, C.; Shan, A. Bioactivity and Bactericidal Mechanism of Histidine-Rich β-Hairpin Peptide Against Gram-Negative Bacteria. Int. J. Mol. Sci. 2019, 20, 3954. https://doi.org/10.3390/ijms20163954
Dong N, Wang C, Zhang T, Zhang L, Xue C, Feng X, Bi C, Shan A. Bioactivity and Bactericidal Mechanism of Histidine-Rich β-Hairpin Peptide Against Gram-Negative Bacteria. International Journal of Molecular Sciences. 2019; 20(16):3954. https://doi.org/10.3390/ijms20163954
Chicago/Turabian StyleDong, Na, Chensi Wang, Tingting Zhang, Lei Zhang, Chenyu Xue, Xinjun Feng, Chongpeng Bi, and Anshan Shan. 2019. "Bioactivity and Bactericidal Mechanism of Histidine-Rich β-Hairpin Peptide Against Gram-Negative Bacteria" International Journal of Molecular Sciences 20, no. 16: 3954. https://doi.org/10.3390/ijms20163954