Characterization of Chemically-Induced Bacterial Ghosts (BGs) Using Sodium Hydroxide-Induced Vibrio parahaemolyticus Ghosts (VPGs)
Abstract
:1. Introduction
2. Results and Discussion
2.1. Effects of Chemicals on Bacterial Cell Envelopes
2.2. Determination of DNA-Free Vibrio parahaemolyticus Ghosts (VPGs) by Agarose Gel Electrophoresis and qPCR
2.3. Analysis of Lipopolysaccharides (LPS) Profile in Chemically-Induced VPGs
2.4. Comparison of Cytotoxicity Tests for VPGs
2.5. Induction of Cytokine Gene Expression in Murine Macrophages-Exposed VPGs
2.6. Morphological Observation of NaOH-Induced VPGs by Scanning Electron Microscopy (SEM)
2.7. Analysis of Protein and DNA Profiles in Sodium Hydroxide-Induced VPGs
3. Materials and Methods
3.1. Bacterial Strain and Culture Condition
3.2. Chemical Agents and Determination of Their MICs (Minimum Inhibitory Concentration)
3.3. Production of VPGs
3.4. Analysis of DNA-Free VPGs by Real-Time PCR
3.5. LPS Extraction, SDS-PAGE, and LAL Assay
3.6. Assessment of Macrophage-Mediated Cytotoxicity
3.7. Quantitative Analysis of Cytokine mRNA by Reverse Transcription (RT)-qPCR
3.8. SEM, SDS-PAGE, and Agarose Gel Electrophoresis Analyses
3.9. Statistical Analysis
4. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Chemical | MIC (mg/mL) | Medium pH |
---|---|---|
Sodium hydroxide (NaOH) | 3.125 | 9.99 |
Acetic acid (CH3COOH) | 6.25 | 4.28 |
Boric acid (BH3O3) | <50 | - |
Citric acid (C6H8O7) | 25 | 3.11 |
Hydrochloric acid (HCl) | 1.56 | 3.95 |
Maleic acid (C4H4O4) | 6.25 | 3.56 |
Sulfuric acid (H2SO4) | 0.781 | 5.83 |
Gene | Orientation | Primer Sequences (5′–3′) |
---|---|---|
16s rRNA | forward | ATATGCCTAGGTGGGATTAGCTAGT |
reverse | TGTCTCAGTTCCAGTGTGGCTG | |
TNF-α | forward | ATGAGCACAGAA AGCATGATCCG |
reverse | GCTGAGACATAGGCACCGC | |
Il-1β | forward | ATGGCAACTGTTCCTGAACTCAACT |
reverse | AGTAGCCCTTCATCTTTTGGGG | |
IL-6 | forward | ATGAAGTTCCTCTCTGCAAGAGACT |
reverse | GTCTCCTCTCCGGACTTGTGA | |
IL-10 | forward | ATGCCTGGCTCAGCACTGCTA |
reverse | CTGGGAAGTGGGTGCAGTTATTG | |
IL-12 | forward | ATGTGTCAATCACGCTACCTCCT |
reverse | GACTGGCTAAGACACCTGGC | |
iNOS | forward | ATGAACCCCAAGAGTTTGACCAGA |
reverse | GGAGCCATAATACTGGTTGATGAAC | |
GAPDH | forward | ATGGTGAAGGTCGGTGTGAACG |
reverse | CAATGAAGGGGTCGTTGATGGC |
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Park, H.J.; Oh, S.; Vinod, N.; Ji, S.; Noh, H.B.; Koo, J.M.; Lee, S.H.; Kim, S.C.; Lee, K.-S.; Choi, C.W. Characterization of Chemically-Induced Bacterial Ghosts (BGs) Using Sodium Hydroxide-Induced Vibrio parahaemolyticus Ghosts (VPGs). Int. J. Mol. Sci. 2016, 17, 1904. https://doi.org/10.3390/ijms17111904
Park HJ, Oh S, Vinod N, Ji S, Noh HB, Koo JM, Lee SH, Kim SC, Lee K-S, Choi CW. Characterization of Chemically-Induced Bacterial Ghosts (BGs) Using Sodium Hydroxide-Induced Vibrio parahaemolyticus Ghosts (VPGs). International Journal of Molecular Sciences. 2016; 17(11):1904. https://doi.org/10.3390/ijms17111904
Chicago/Turabian StylePark, Hyun Jung, Sung Oh, Nagarajan Vinod, Seongmi Ji, Han Byul Noh, Jung Mo Koo, Su Hyeong Lee, Sei Chang Kim, Ki-Sung Lee, and Chang Won Choi. 2016. "Characterization of Chemically-Induced Bacterial Ghosts (BGs) Using Sodium Hydroxide-Induced Vibrio parahaemolyticus Ghosts (VPGs)" International Journal of Molecular Sciences 17, no. 11: 1904. https://doi.org/10.3390/ijms17111904