Abstract
Arenicin-1, a 21-mer antimicrobial peptide exerts significant broad-spectrum antimicrobial activity with membrane-active mechanisms. However, owing to multiple mechanisms of cell death, the antibacterial mechanism of arenicin-1 requires detailed analysis. In the present study, arenicin-1-treated bacteria underwent an apoptosis-like response, which was mechanistically and morphologically similar to eukaryotic apoptosis. Changes in the physiological status of arenicin-1-treated bacterial cells involved accumulation of reactive oxygen species, imbalance of intracellular calcium gradients, disruption of membrane potential, bacterial caspase-like protein activation, and DNA damage. In arenicin-1-induced apoptosis-like death, autocleavage of LexA was observed because of the activation of the caspase-like activity of RecA. Additionally, typical reactive oxygen species such as superoxide, hydrogen peroxide, and hydroxyl radicals, were scavenged in arenicin-1-treated cells to assess the role of specific reactive oxygen species. Scavenging of hydrogen peroxide interfered with the activity of arenicin-1 in Escherichia coli, whereas the superoxide and hydroxyl radicals level did not affect arenicin-1-induced apoptosis-like death activity. Furthermore, inhibition of Fenton reaction attenuated apoptosis-like response. In conclusion, arenicin-1-induced apoptosis like death requires SOS response proteins and is mediated by hydrogen peroxide and Fenton reaction in E. coli. Arenicin-1 may be a representative antimicrobial peptide with potent apoptotic response against E. coli.
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Abbreviations
- PCD:
-
Programmed cell death
- TLD:
-
Thymine-less death
- ALD:
-
Apoptotic-like death
- AMP:
-
Antimicrobial peptides
- PBS:
-
Phosphate-buffered saline
- GSH:
-
Reduced glutathione
- DiBAC4(3):
-
Bis-(1,3-dibutylbarbituric acid) trimethine oxonol
- H2DCFDA:
-
2′,7′-Dichlorodihydrofluorescein diacetate
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Acknowledgements
This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korean government (MSIP) (No. 2017R1A2B4005811).
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294_2018_855_MOESM1_ESM.tif
Fig. S1. Oxidative stress in ΔRecA and K996 cells treated with arenicin-1. ΔRecA cells were treated with arenicin-1 (12.5 µM) or norfloxacin (2.3 µM). K996 cells were treated with arenicin-1 (12.5 µM) or norfloxacin (2.3 µM). (A) ROS production was detected via H2DCFDA staining (B) Decrease in GSH levels indicate oxidative damage. (C) Increase in MDA levels indicate lipid peroxidation. The data represents the average, standard deviation, and p values from three independent experiments (*p < 0.1; **p < 0.05; ***p < 0.01 vs the untreated; Student’s t test) (TIF 10063 KB)
294_2018_855_MOESM2_ESM.tif
Fig. S2. Features of apoptotic-like death in ΔRecA and K996 cells induced by arenicin-1. Analysis of membrane depolarization in ΔRecA and K996 cells in the presence/absence of arenicin-1 and ROS scavenger. Flow cytometric analysis of membrane depolarization using DiBAC4(3) (TIF 12006 KB)
294_2018_855_MOESM3_ESM.tif
Fig. S3 Features of apoptotic-like death in ΔRecA and K996 cells induced by arenicin-1. Analysis of cell filamentation in ΔRecA and K996 cells treated with/without arenicin-1 and ROS scavenger. Induction-mediated cell size changes. An increase in FS and SS indicated cell filamentation (TIF 13952 KB)
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Lee, H., Lee, D.G. Arenicin-1-induced apoptosis-like response requires RecA activation and hydrogen peroxide against Escherichia coli. Curr Genet 65, 167–177 (2019). https://doi.org/10.1007/s00294-018-0855-3
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DOI: https://doi.org/10.1007/s00294-018-0855-3