Dual Antibacterial Activities and Biofilm Eradication of a Marine Peptide-N6NH2 and Its Analogs against Multidrug-Resistant Aeromonas veronii
Abstract
:1. Introduction
2. Results
2.1. Design and Physicochemical Properties of N6NH2 and Its Analogs
2.2. N6NH2 and Its Analogs Showed Potent Antimicrobial Activity
2.3. N6NH2 and Its Analogs Showed Additive Effects with Antibiotics and Greater Post-Antibiotic Effect (PAE) Values
2.4. DN6NH2 and N6PNH2 Had Higher Stability in Different Conditions
2.5. All Analogs Exhibited No or Low Toxicity and Resistance
2.6. Antibacterial Mechanism of N6NH2 and Its Analogs
2.6.1. N6NH2 and Its Analogs Permeabilized the Outer Cell Membranes and Disrupted the Membrane Potentials
2.6.2. Peptides Exposure Affects Adenosine Triphosphate (ATP) Release from Cells
2.6.3. All Analogs of N6NH2 Bound to Bacterial Genomic DNA and Changed DNA Structure
2.7. All Analogs of N6NH2 Induced Morphological Changes in A. veronii
2.7.1. Scanning Electron Microscope (SEM) Observations
2.7.2. Transmission Electron Microscope (TEM) and Confocal Laser Scanning Microscope (CLSM) Observations
2.8. N6NH2 and Its Analogs Eliminated MDR A. veronii Biofilms and Persisters
2.8.1. Inhibition of Biofilm Formation
2.8.2. Eradication of Mature Biofilms
2.8.3. Killing Persisters in Biofilm
2.9. N6NH2 and Its Analogs Protected Mice from Catheter-Associated Biofilm Infection with MDR A. veronii
2.9.1. Protection of Biofilm-Infected Mice
2.9.2. Protection of Tissues from A. veronii ACCC61732 Biofilm
2.10. N6NH2 and Its Analogs Protected Mice from Bacterial Infection with MDR A. veronii
2.10.1. Protection of Mice
2.10.2. Inhibition of Bacterial Translocation
2.10.3. Regulation of Cytokines
2.10.4. Alleviation of the Organ Injury
3. Discussion
4. Materials and Methods
4.1. Reagents, Cell Lines, and Model Animals
4.2. Physiochemical Properties of N6NH2 and Its Analogs
4.3. Antibacterial Activities and Time-Killing Curves of N6NH2 and Its Analogs
4.4. Synergism and PAE of N6NH2 and Its Analogs against A. veronii
4.5. Stability of N6NH2 and Its Analogs
4.5.1. Temperature, pH, Salt, and Enzyme Sensitivity
4.5.2. Stability in Gastric/Intestinal Fluid and Serum
4.6. Hemolysis, Cytotoxicity, and Resistance of N6NH2 and Its Analogs
4.6.1. Hemolysis
4.6.2. Cytotoxicity
4.6.3. Resistance
4.7. Mechanism of N6NH2 and Its Analogs
4.7.1. Effects on the Cell Membrane and Membrane Potential
4.7.2. Measure of ATP Release
4.7.3. Effects on Bacterial Genomic DNA
4.7.4. Effects of N6NH2 and Its Analogs on Bacterial Morphology
4.8. Effects of N6NH2 and Its Analogs on Biofilms and Persisters of MDR A. veronii
4.8.1. Early and Mature Biofilms
4.8.2. Persisters in Biofilms
4.9. Efficacy of N6NH2 and Its Analogs in a Mouse Model of Catheter-Associated Biofilm Infection
4.9.1. Effects on Catheter-Associated Biofilms
4.9.2. Effects on Skin Ulcer in the Mouse Back
4.10. Efficacy of N6NH2 and Its Analogs in a Mouse Peritonitis Model
4.10.1. Survival of Mice
4.10.2. Effects on Bacterial Translocation and Cytokines
4.10.3. Effects on the Injury of Multiple Organs
4.11. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
CIP | ciprofloxacin |
AMP | antimicrobial peptide(s) |
Orn | ornithine |
MDR | multidrug resistant |
MIC | minimum inhibitory concentration |
MW | molecular weight |
MS | mass spectroscopy |
GRAVY | grand average of hydropathicity |
AI | aliphatic index |
BI | boman index |
pI | isoelectric point |
FICI | fractional inhibitory concentration index |
PAE | postantibiotic effect |
MTT | methylthiazolyldiphenyl-tetrazolium |
SGF | simulated gastric fluid |
SIF | simulated intestinal fluid |
GEN | gentamicin |
NPN | n-phenyl-1-naphthylamine |
PI | propidium iodide |
DISC3(5)) | 3,3′-dipropylthiadicarbocyanine iodide |
ATP | adenosine triphosphate |
CD | circular dichroism |
SEM | scanning electron microscope |
OMVs | outer membrane vesicles |
PCR | polymerase chain reaction |
TEM | transmission electron microscope |
CLSM | confocal laser scanning microscope |
TNF | tumor necrosis factor |
IL | interleukin |
ELISA | enzyme-linked immunosorbent assay |
HEPES | n-2-hydroxyethylpiperazine-n-2-ethane sulfonic acid |
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Peptides | Amino Acid Sequences | Length | Theoretical MW (Da) | Measured MW (Da) | Charge (+) | GRAVY | AI | BI (kcal mol-1) | pI | Hydrophobicity | AH % | Extinction Coefficient (M−1 cm−1) | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
N6NH2 | GFAWNVCVYRNGVRVCHRRAN-NH2 | 21 | 2476.85 | 2476.8 | 5 | NP | NP | NP | 11.64 | 0.375 | NP | 6970 | |
DN6NH2 | GfawnvcvyrnGvrvchrran-NH2 | 21 | 2476.85 | 2474.88 | 5 | NP | NP | NP | 11.64 | 0.375 | NP | 6970 | |
N6PNH2 | GFAWNVCVYRNGVRVCHRPAN-NH2 | 21 | 2417.78 | 2415.81 | 4 | NP | NP | NP | 10.91 | 0.457 | NP | 6970 | |
V112N6NH2 | VFAWNVCVYRNVVRVCHRRAN-NH2 | 21 | 2561.01 | 2559.04 | 5 | NP | NP | NP | 11.64 | 0.491 | NP | 6970 | |
Guo-N6NH2 | Gu-OGFAWNVCVYRNGVRVCHRRAN-NH2 | 21 | NP | 2631.08 | 6 | NP | NP | NP | NP | NP | NP | NP | |
N2143 | VCVYRGFAWNCHRRANNGVRV | 21 | 2477.85 | 2475.81 | 4 | −0.31 | 64.76 | 2.61 | 10.72 | 0.375 | 23.81% | 6970 | |
N2413 | VCVYRCHRRANGFAWNNGVRV | 21 | 2477.85 | 2475.81 | 4 | −0.31 | 64.76 | 2.61 | 10.72 | 0.375 | 0.00% | 6970 | |
SN1 | AACCGFWNVVYRNGVRVHRRN | 21 | 2477.85 | 2477.83 | 4 | −0.31 | 64.76 | 2.61 | 10.72 | 0.375 | 0.00% | 6970 | |
SN3 | GFWNVVYRNGVAACCRVHRRN | 21 | 2477.85 | 2477.83 | 4 | −0.31 | 64.76 | 2.61 | 10.72 | 0.375 | 33.33% | 6970 |
Species and Strains | MIC | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
N6NH2 | DN6NH2 | N6PNH2 | V112N6NH2 | Guo-N6NH2 | CIP | |||||||
μg/mL | μM | μg/mL | μM | μg/mL | μM | μg/mL | μM | μg/mL | μM | μg/mL | μM | |
Gram-negative bacteria | ||||||||||||
Aeromonas veronii ACCC61732 a | 4 | 1.61 | 4 | 1.62 | 16 | 6.62 | 16 | 6.25 | 8 | 3.04 | 0.125 | 0.38 |
A. veronii X-1-06909 b | 32 | 12.9 | 32 | 12.93 | 64 | 26.49 | 32 | 12.51 | 16 | 6.08 | 0.25 | 0.75 |
A. veronii CL0901 b | 16 | 6.46 | 32 | 12.93 | 64 | 26.49 | 32 | 12.51 | 16 | 6.08 | 0.0625 | 0.19 |
A. veronii ATCC35624 | 8 | 3.23 | 64 | 25.86 | 64 | 26.49 | 16 | 6.25 | 32 | 12.16 | <0.0625 | <0.19 |
Escherichia coli CVCC195 | 4 | 1.61 | 8 | 3.23 | 8 | 3.31 | 8 | 3.13 | 4 | 1.52 | <0.0625 | <0.19 |
E. coli CVCC1515 | 2 | 0.81 | 4 | 1.62 | 4 | 1.66 | 4 | 1.56 | 2 | 0.76 | <0.0625 | <0.19 |
E. coli CVCC25922 | 4 | 1.61 | 8 | 3.23 | 8 | 3.31 | 8 | 3.13 | 4 | 1.52 | <0.0625 | <0.19 |
E. coli CVCCO157 | 4 | 1.61 | 4 | 1.62 | 8 | 3.31 | 8 | 3.13 | 4 | 1.52 | <0.0625 | <0.19 |
Salmonella typhimurium ATCC14028 | 4 | 1.61 | 8 | 3.23 | 16 | 6.62 | 16 | 6.25 | 4 | 1.52 | 0.0625 | 0.19 |
S. pullorum CVCC1809 | 4 | 1.61 | 8 | 3.23 | 8 | 3.31 | 8 | 3.13 | 4 | 1.52 | <0.0625 | <0.19 |
S. pullorum CVCC1789 | 8 | 3.23 | 16 | 6.46 | 16 | 6.62 | 16 | 6.25 | 8 | 3.04 | <0.0625 | <0.19 |
S. pullorum CVCC533 | 4 | 1.61 | 16 | 6.46 | 32 | 13.25 | 16 | 6.25 | 8 | 3.04 | <0.0625 | <0.19 |
S. enteritidis CVCC3377 | 2 | 0.81 | 2 | 0.81 | 2 | 0.83 | 2 | 0.78 | 2 | 0.76 | <0.0625 | <0.19 |
Pseudomonas aeruginosa CICC21630 | 64 | 25.8 | 8 | 3.23 | >64 | >26.49 | 32 | 12.5 | >64 | >24.3 | 0.125 | 0.38 |
Gram-positive bacteria | ||||||||||||
Staphylococcus aureus ATCC43300 | 16 | 6.46 | 4 | 1.62 | >64 | >26.49 | 32 | 12.5 | 16 | 6.08 | 0.25 | 0.75 |
S. aureus ATCC546 | 16 | 6.46 | 16 | 6.46 | >64 | >26.49 | 16 | 6.25 | 16 | 6.08 | 0.125 | 0.38 |
S. aureus ATCC25923 | 32 | 12.9 | 8 | 3.23 | >64 | >26.49 | 32 | 12.5 | 32 | 12.16 | 0.125 | 0.38 |
S. hyicus NCTC10350 | 32 | 12.9 | 8 | 3.23 | >128 | >52.98 | 64 | 25.01 | 64 | 24.32 | 0.125 | 0.38 |
S. hyicus 437-2 c | 32 | 12.9 | 8 | 3.23 | >64 | >26.49 | 32 | 12.5 | 32 | 12.16 | 8 | 24.14 |
Fungus | ||||||||||||
Candida albicans CMCC98001 | >128 | >52 | 128 | 51.72 | >128 | >52.98 | >128 | >50.02 | >128 | >48.6 | >128 | >386.3 |
Peptides | PAE (h) | ||
---|---|---|---|
1× MIC | 2× MIC | 4× MIC | |
N6NH2 | 0.69 ± 0.01 | 0.7 ± 0.01 | 1.17 ± 0.22 |
DN6NH2 | 0.68 ± 0.01 | 1.13 ± 0.11 | 3.36 ± 0.19 |
N6PNH2 | 0.52 ± 0.01 | 0.62 ± 0.01 | 0.68 ± 0.01 |
V112N6NH2 | 0.74 ± 0.02 | 1.48 ± 0.05 | 2.15 ± 0.13 |
Guo-N6NH2 | 0.62 ± 0.01 | 1.32 ± 0.08 | 2.07 ± 0.08 |
CIP | 0.6 ± 0.01 | 0.63 ± 0.1 | 0.67 ± 0.02 |
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Li, T.; Wang, Z.; Han, H.; Teng, D.; Mao, R.; Hao, Y.; Yang, N.; Wang, X.; Wang, J. Dual Antibacterial Activities and Biofilm Eradication of a Marine Peptide-N6NH2 and Its Analogs against Multidrug-Resistant Aeromonas veronii. Int. J. Mol. Sci. 2020, 21, 9637. https://doi.org/10.3390/ijms21249637
Li T, Wang Z, Han H, Teng D, Mao R, Hao Y, Yang N, Wang X, Wang J. Dual Antibacterial Activities and Biofilm Eradication of a Marine Peptide-N6NH2 and Its Analogs against Multidrug-Resistant Aeromonas veronii. International Journal of Molecular Sciences. 2020; 21(24):9637. https://doi.org/10.3390/ijms21249637
Chicago/Turabian StyleLi, Ting, Zhenlong Wang, Huihui Han, Da Teng, Ruoyu Mao, Ya Hao, Na Yang, Xiumin Wang, and Jianhua Wang. 2020. "Dual Antibacterial Activities and Biofilm Eradication of a Marine Peptide-N6NH2 and Its Analogs against Multidrug-Resistant Aeromonas veronii" International Journal of Molecular Sciences 21, no. 24: 9637. https://doi.org/10.3390/ijms21249637