Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Antibacterial Mode of Action of β-Amyrin Promotes Apoptosis-Like Death in Escherichia coli by Producing Reactive Oxygen Species

  • Giyeol Han (School of Life Sciences, BK 21 FOUR KNU Creative BioResearch Group, Kyungpook National University) ;
  • Dong Gun Lee (School of Life Sciences, BK 21 FOUR KNU Creative BioResearch Group, Kyungpook National University)
  • Received : 2022.09.26
  • Accepted : 2022.11.02
  • Published : 2022.12.28
Download PDF
⟨ Previous Next ⟩

Abstract

β-Amyrin is a pentacyclic triterpene widely distributed in leaves and stems worldwide. The ability of β-amyrin to induce the production of reactive oxygen species (ROS) in microorganisms suggests its potential as an antimicrobial agent. Thus, this study aimed to elucidate the antibacterial mode of action of β-amyrin. We treated Escherichia coli cells with β-amyrin and found that it triggered ROS accumulation. Excessive stress caused by ROS, particularly hydroxyl radicals, induces glutathione (GSH) dysfunction. GSH protects cells from oxidative and osmotic stresses; thus, its dysfunction leads to membrane depolarization. The resultant change in membrane potential leads to the release of apoptotic proteins, such as caspases. The activated caspases-like protein promotes the cleavage of DNA into single strands, which is a hallmark of apoptosis-like death in bacteria. Apoptotic cells usually undergo events such as DNA fragmentation and phosphatidylserine exposure, differentiating them from necrotic cells, and the cells treated with β-amyrin in this study were positive for annexin V and negative for propidium iodide, indicating apoptosis-like death. In conclusion, our findings suggest that the antibacterial mode of action of β-amyrin involves the induction of ROS, which resulted in apoptosis-like death in E. coli.

Keywords

Acknowledgement

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2022R1A2C1011642).

References

  1. Jager S, H Trojan, T Kopp, MN Laszczyk, A Scheffler. 2009. Pentacyclic triterpene distribution in various plants-rich sources for a new group of multi-potent plant extracts. Molecules 14: 2016-2031. https://doi.org/10.3390/molecules14062016
  2. Cheng Z, Y Li, X Zhu, K Wang, Y Ali, W Shu, et al. 2021. The potential application of pentacyclic triterpenoids in the prevention and treatment of retinal diseases. Planta Med. 87: 511-527. https://doi.org/10.1055/a-1377-2596
  3. Ghiulai R, OJ Rosca, DS Antal, M Mioc, A Mioc, R Racoviceanu, et al., 2020. Tetracyclic and pentacyclic triterpenes with high therapeutic efficiency in wound healing approaches. Molecules 25: 5557.
  4. Wu P, B Tu, J Liang, S Guo, N Cao, S Chen, et al. 2021. Synthesis and biological evaluation of pentacyclic triterpenoid derivatives as potential novel antibacterial agents. Bioorg. Chem. 109: 104692.
  5. Kwun MS, HJ Lee, DG Lee, 2021. β-amyrin-induced apoptosis in Candida albicans triggered by calcium. Fungal Biol. 125: 630-636. https://doi.org/10.1016/j.funbio.2021.03.006
  6. Boar RB, J Allen. 1973. β-Amyrin triterpenoids. Phytochemistry 12: 2571-2578. https://doi.org/10.1016/0031-9422(73)85059-9
  7. Melo CM, KMMB Carvalho, JC de Sousa Neves, TC Morais, VS Rao, FA Santos, et al. 2010. α, β-amyrin, a natural triterpenoid ameliorates L-arginine-induced acute pancreatitis in rats. World J. Gastroenterol. 16: 4272-4280. https://doi.org/10.3748/wjg.v16.i34.4272
  8. Zhao X, K Drlica. 2014. Reactive oxygen species and the bacterial response to lethal stress. Curr. Opin. Microbiol. 21: 1-6. https://doi.org/10.1016/j.mib.2014.06.008
  9. Fasnacht M, N Polacek. 2021. Oxidative stress in bacteria and the central dogma of molecular biology. Front. Mol. Biosci. 8: 671037.
  10. Haanen C, I Vermes. 1995. Apoptosis and inflammation. Mediat. Inflamm. 4: 5-15. https://doi.org/10.1155/S0962935195000020
  11. Kim H, DG Lee. 2020. Nitric oxide-inducing genistein elicits apoptosis-like death via an intense SOS response in Escherichia coli. Appl. Microbiol. Biotechnol.104: 10711-10724. https://doi.org/10.1007/s00253-020-11003-1
  12. Okoye NN, DL Ajaghaku, HN Okeke, EE Ilodigwe, CS Nworu, FBC Okoye. 2014. beta-Amyrin and alpha-amyrin acetate isolated from the stem bark of Alstonia boonei display profound anti-inflammatory activity. Pharm. Biol. 52:1478-1486. https://doi.org/10.3109/13880209.2014.898078
  13. Elmore S 2007. Apoptosis: a review of programmed cell death. Toxicol. Pathol. 35: 495-516. https://doi.org/10.1080/01926230701320337
  14. Kwon DH, H-J Cha, H Lee, S-H Hong, C Park, S-H Park, et al. 2019. Protective effect of glutathione against oxidative stress-induced cytotoxicity in RAW 264.7 macrophages through activating the nuclear factor erythroid 2-related factor-2/heme oxygenase-1 pathway. Antioxidants 8: 82.
  15. Rahman I, A Kode, SK Biswas. 2019. Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method. Nat. Protoc.1: 3159-3165. https://doi.org/10.1038/nprot.2006.378
  16. Goldstein EJ. 1987. Norfloxacin, a fluoroquinolone antibacterial agent: classification, mechanism of action, and in vitro activity. Am. J. Med. 82: 3-17. https://doi.org/10.1016/0002-9343(87)90612-7
  17. Suski JM, M Lebiedzinska, M Bonora, P Pinton, J Duszynski, MR Wieckowski. 2012. Relation between mitochondrial membrane potential and ROS formation, in Mitochondrial bioenergetics. pp. 183-205. 2012, Springer.
  18. Moungjaroen J, U Nimmannit, PS Callery, L Wang, N Azad, V Lipipun, et al., 2006. Reactive oxygen species mediate caspase activation and apoptosis induced by lipoic acid in human lung epithelial cancer cells through Bcl-2 down-regulation. J. Pharmacol. Exp. Ther. 319: 1062-1069. https://doi.org/10.1124/jpet.106.110965
  19. Edgington-Mitchell, LE, M Bogyo, Detection of active caspases during apoptosis using fluorescent activity-based probes, in Programmed Cell Death. pp. 27-39. 2016, Springer.
  20. Bortner CD, NB Oldenburg, JA Cidlowski. 1995. The role of DNA fragmentation in apoptosis. Trends Cell Biol. 5: 21-26. https://doi.org/10.1016/S0962-8924(00)88932-1
  21. Janicke RU, ML Sprengart, MR Wati, AG Porter. 1998. Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J. Biol. Chem. 273: 9357-9360. https://doi.org/10.1074/jbc.273.16.9357
  22. Kyrylkova K, S Kyryachenko, M Leid, C Kioussi. 2012. Detection of apoptosis by TUNEL assay, in Odontogenesis. pp. 41-47. Springer.
  23. Saraste A, K Pulkki. 2000. Morphologic and biochemical hallmarks of apoptosis. Cardiovasc. Res. 45: 528-537. https://doi.org/10.1016/S0008-6363(99)00384-3
  24. Marino, G, G Kroemer. 2013. Mechanisms of apoptotic phosphatidylserine exposure. Cell Res. 23: 1247-1248. https://doi.org/10.1038/cr.2013.115
  25. Crowley LC, BJ Marfell, AP Scott, NJ Waterhouse. 2016. Quantitation of apoptosis and necrosis by annexin V binding, propidium iodide uptake, and flow cytometry. Cold Spring Harb. Protoc. 2016. doi: 10.1101/pdb.prot087288..
  26. Safayhi H, E-R Sailer. 1997. Anti-inflammatory actions of pentacyclic triterpenes. Planta Med. 63: 487-493. https://doi.org/10.1055/s-2006-957748
  27. Hernandez-Vazquez, L, J Palazon Barandela, A Navarro-Ocana. 2012. The pentacyclic triterpenes α, β-amyrins: a review of sources and biological activities. Chapter 23 in: Rao, Venketeshwer. Phytochemicals: A Global Perspective of Their Role in Nutrition and Health. IntechOpen. ISBN: 978-953-51-4317-8. DOI: 10.5772/1387 pp. 487-502., 2012.
  28. Memar MY, R Ghotaslou, M Samiei, K Adibkia. 2018. Antimicrobial use of reactive oxygen therapy: current insights. Infect. Drug Resist. 11: 567-576. https://doi.org/10.2147/IDR.S142397
  29. Ezraty B, A Gennaris, F Barras, J-F Collet. 2017. Oxidative stress, protein damage and repair in bacteria. Nat. Rev. Microbiol. 15: 385-396. https://doi.org/10.1038/nrmicro.2017.26
  30. Brookes PS, Y Yoon, JL Robotham, M Anders, S-S Sheu. 2004. Calcium, ATP, and ROS: a mitochondrial love-hate triangle. Am. J. Physiol. Cell Physiol. 287: C817-833. https://doi.org/10.1152/ajpcell.00139.2004
  31. Sheng H, K Nakamura, T Kanno, K Sasaki, Y Niwano. 2015. Microbicidal activity of artificially generated hydroxyl radicals, pp. 203-215. in Interface Oral Health Science 2014. Springer, Tokyo.
  32. Masip L, K Veeravalli, G Georgiou. 2006. The many faces of glutathione in bacteria. Antioxid. Redox Signal. 8: 753-762. https://doi.org/10.1089/ars.2006.8.753
  33. Hong Y, J Zeng, X Wang, K Drlica, X Zhao. 2019. Post-stress bacterial cell death mediated by reactive oxygen species. Proc. Natl. Acad. Sci. USA 116: 10064-10071. https://doi.org/10.1073/pnas.1901730116
  34. Te Winkel, JD, DA Gray, KH Seistrup, LW Hamoen, H Strahl. 2016. Analysis of antimicrobial-triggered membrane depolarization using voltage sensitive dyes. Front. Cell Dev. Biol. 4: 29.
  35. Gough NR. 2011. Stressing bacteria to death. Sci. Signal. 4: ec164.
  36. Asplund-Samuelsson J. 2015. The art of destruction: revealing the proteolytic capacity of bacterial caspase homologs. Mol. Microbiol. 98: 1-6. https://doi.org/10.1111/mmi.13111
  37. Bayles KW. 2014. Bacterial programmed cell death: making sense of a paradox. Nat. Rev. Microbiol. 12: 63-69. https://doi.org/10.1038/nrmicro3136
  38. Shlomovitz I, M Speir, M Gerlic. 2019. Flipping the dogma-phosphatidylserine in non-apoptotic cell death. Cell Commun. Signal. 17: 139.