Poly-γ-Glutamic Acid from a Novel Bacillus subtilis Strain: Strengthening the Skin Barrier and Improving Moisture Retention in Keratinocytes and a Reconstructed Skin Model
Abstract
1. Introduction
2. Results and Discussion
2.1. Isolation and Identification
2.2. Identification of γ-PGA
2.3. Cytotoxicity of γ-PGA in Keratinocytes
2.4. Effect of γ-PGA on Physical Skin Barrier-Related Markers in Keratinocytes
2.5. Effect of γ-PGA on Permeability Skin Barrier-Related Markers in Keratinocytes
2.6. Effect of γ-PGA on Hyaluronic Acid Synthesis in Keratinocytes
2.7. Effect of γ-PGA on AQP3 Expression in Keratinocytes
2.8. Effect of γ-PGA on Skin Barrier-Related Markers in a Reconstructed Skin Model
3. Materials and Methods
3.1. Bacterial Strain Isolation and Identification
3.2. Microbial Culture and Reagent Preparation
3.3. Compound Isolation and Identification
3.4. Cell Culture
3.5. Cell Viability
3.6. RT-PCR
3.7. Immunocytofluorescence Analysis
3.8. Reconstructed Skin Model
3.9. Histological Analysis
3.10. Image Acquisition and Analysis
3.11. Statistical Analysis
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
- Awasthi, S.K.; Kumar, M.; Kumar, V.; Sarsaiya, S.; Anerao, P.; Ghosh, P.; Singh, L.; Liu, H.; Zhang, Z.; Awasthi, M.K. A comprehensive review on recent advancements in biodegradation and sustainable management of biopolymers. Environ. Pollut. 2022, 307, 119600. [Google Scholar] [CrossRef] [PubMed]
- Shih, I.-L.; Van, Y.-T. The production of poly-(γ-glutamic acid) from microorganisms and its various applications. Bioresour. Technol. 2001, 79, 207–225. [Google Scholar] [CrossRef]
- Zeng, W.; Liu, Y.; Shu, L.; Guo, Y.; Wang, L.; Liang, Z. Production of ultra-high-molecular-weight poly-γ-glutamic acid by a newly isolated Bacillus subtilis strain and genomic and transcriptomic analyses. Biotechnol. J. 2024, 19, e2300614. [Google Scholar] [CrossRef]
- Isago, Y.; Suzuki, R.; Isono, E.; Noguchi, Y.; Kuroyanagi, Y. Development of a Freeze-Dried Skin Care Product Composed of Hyaluronic Acid and Poly(γ-Glutamic Acid) Containing Bioactive Components for Application after Chemical Peels. Open J. Regen. Med. 2014, 3, 49441. [Google Scholar] [CrossRef][Green Version]
- Wang, X.; Mohammad, I.S.; Fan, L.; Zhao, Z.; Nurunnabi, M.; Sallam, M.A.; Wu, J.; Chen, Z.; Yin, L.; He, W. Delivery strategies of amphotericin B for invasive fungal infections. Acta Pharm. Sin. B 2021, 11, 2585–2604. [Google Scholar] [CrossRef] [PubMed]
- Liu, X.; Liu, F.; Liu, S.; Li, H.; Ling, P.; Zhu, X. Poly-γ-glutamate from Bacillus subtilis inhibits tyrosinase activity and melanogenesis. Appl. Microbiol. Biotechnol. 2013, 97, 9801–9809. [Google Scholar] [CrossRef]
- Vaughn, A.R.; Clark, A.K.; Sivamani, R.K.; Shi, V.Y. Natural Oils for Skin-Barrier Repair: Ancient Compounds Now Backed by Modern Science. Am. J. Clin. Dermatol. 2018, 19, 103–117. [Google Scholar] [CrossRef]
- Almoughrabie, S.; Cau, L.; Cavagnero, K.; O’Neill, A.M.; Li, F.; Roso-Mares, A.; Mainzer, C.; Closs, B.; Kolar, M.J.; Williams, K.J.; et al. Commensal Cutibacterium acnes induce epidermal lipid synthesis important for skin barrier function. Sci. Adv. 2023, 9, eadg6262. [Google Scholar] [CrossRef]
- Ohno, Y.; Kamiyama, N.; Nakamichi, S.; Kihara, A. PNPLA1 is a transacylase essential for the generation of the skin barrier lipid ω-O-acylceramide. Nat. Commun. 2017, 8, 14610. [Google Scholar] [CrossRef] [PubMed]
- Bouwstra, J.A.; Nădăban, A.; Bras, W.; McCabe, C.; Bunge, A.; Gooris, G.S. The skin barrier: An extraordinary interface with an exceptional lipid organization. Prog. Lipid Res. 2023, 92, 101252. [Google Scholar] [CrossRef] [PubMed]
- Fuchs, E. Epidermal differentiation and keratin gene expression. J. Cell Sci. Suppl. 1993, 17, 197–208. [Google Scholar] [CrossRef]
- Furue, M. Regulation of Filaggrin, Loricrin, and Involucrin by IL-4, IL-13, IL-17A, IL-22, AHR, and NRF2: Pathogenic Implications in Atopic Dermatitis. Int. J. Mol. Sci. 2020, 21, 5382. [Google Scholar] [CrossRef]
- Steinert, P.M.; Marekov, L.N. The proteins elafin, filaggrin, keratin intermediate filaments, loricrin, and small proline-rich proteins 1 and 2 are isodipeptide cross-linked components of the human epidermal cornified cell envelope. J. Biol. Chem. 1995, 270, 17702–17711. [Google Scholar] [CrossRef] [PubMed]
- Cork, M.J.; Danby, S.G.; Vasilopoulos, Y.; Hadgraft, J.; Lane, M.E.; Moustafa, M.; Guy, R.H.; Macgowan, A.L.; Tazi-Ahnini, R.; Ward, S.J. Epidermal barrier dysfunction in atopic dermatitis. J. Investig. Dermatol. 2009, 129, 1892–1908. [Google Scholar] [CrossRef] [PubMed]
- Harding, C.R.; Watkinson, A.; Rawlings, A.V.; Scott, I.R. Dry skin, moisturization and corneodesmolysis. Int. J. Cosmet. Sci. 2000, 22, 21–52. [Google Scholar] [CrossRef] [PubMed]
- Dübe, B.; Lüke, H.J.; Aumailley, M.; Prehm, P. Hyaluronan reduces migration and proliferation in CHO cells. Biochim. Biophys. Acta 2001, 1538, 283–289. [Google Scholar] [CrossRef]
- Song, H.; Jin, M.; Lee, S. Effect of Ferulic Acid Isolated from Cnidium Officinale on the Synthesis of Hyaluronic Acid. J. Soc. Cosmet. Sci. Korea 2013, 39, 281–288. [Google Scholar] [CrossRef]
- Bollag, W.B.; Aitkens, L.; White, J.; Hyndman, K.A. Aquaporin-3 in the epidermis: More than skin deep. Am. J. Physiol. Cell Physiol. 2020, 318, C1144–C1153. [Google Scholar] [CrossRef]
- Boury-Jamot, M.; Sougrat, R.; Tailhardat, M.; Le Varlet, B.; Bonté, F.; Dumas, M.; Verbavatz, J.M. Expression and function of aquaporins in human skin: Is aquaporin-3 just a glycerol transporter? Biochim. Biophys. Acta 2006, 1758, 1034–1042. [Google Scholar] [CrossRef] [PubMed]
- Qin, H.; Zheng, X.; Zhong, X.; Shetty, A.K.; Elias, P.M.; Bollag, W.B. Aquaporin-3 in keratinocytes and skin: Its role and interaction with phospholipase D2. Arch. Biochem. Biophys. 2011, 508, 138–143. [Google Scholar] [CrossRef] [PubMed]
- Kim, N.H.; Lee, A.Y. Reduced aquaporin3 expression and survival of keratinocytes in the depigmented epidermis of vitiligo. J. Investig. Dermatol. 2010, 130, 2231–2239. [Google Scholar] [CrossRef] [PubMed]
- Ikarashi, N.; Ogiue, N.; Toyoda, E.; Kon, R.; Ishii, M.; Toda, T.; Aburada, T.; Ochiai, W.; Sugiyama, K. Gypsum fibrosum and its major component CaSO4 increase cutaneous aquaporin-3 expression levels. J. Ethnopharmacol. 2012, 139, 409–413. [Google Scholar] [CrossRef] [PubMed]
- Ikarashi, N.; Kon, R.; Kaneko, M.; Mizukami, N.; Kusunoki, Y.; Sugiyama, K. Relationship between Aging-Related Skin Dryness and Aquaporins. Int. J. Mol. Sci. 2017, 18, 1559. [Google Scholar] [CrossRef] [PubMed]
- Filatov, V.; Sokolova, A.; Savitskaya, N.; Olkhovskaya, M.; Varava, A.; Ilin, E.; Patronova, E. Synergetic Effects of Aloe Vera Extract with Trimethylglycine for Targeted Aquaporin 3 Regulation and Long-Term Skin Hydration. Molecules 2024, 29, 1540. [Google Scholar] [CrossRef]
- Suhail, S.; Sardashti, N.; Jaiswal, D.; Rudraiah, S.; Misra, M.; Kumbar, S.G. Engineered Skin Tissue Equivalents for Product Evaluation and Therapeutic Applications. Biotechnol. J. 2019, 14, e1900022. [Google Scholar] [CrossRef]
- Lelièvre, D.; Canivet, F.; Thillou, F.; Tricaud, C.; Le Floc’h, C.; Bernerd, F. Use of reconstructed skin model to assess the photoprotection afforded by three sunscreen products having different SPF values against DNA lesions and cellular alterations. J. Photochem. Photobiol. 2024, 19, 100213. [Google Scholar] [CrossRef]
- Plaza, C.; Meyrignac, C.; Botto, J.M.; Capallere, C. Characterization of a New Full-Thickness In Vitro Skin Model. Tissue Eng. Part C Methods 2021, 27, 411–420. [Google Scholar] [CrossRef]
- Hall, M.J.; Lopes-Ventura, S.; Neto, M.V.; Charneca, J.; Zoio, P.; Seabra, M.C.; Oliva, A.; Barral, D.C. Reconstructed human pigmented skin/epidermis models achieve epidermal pigmentation through melanocore transfer. Pigment. Cell Melanoma Res. 2022, 35, 425–435. [Google Scholar] [CrossRef] [PubMed]
- Ryu, J.S.; Park, H.S.; Kim, M.J.; Lee, J.; Jeong, E.Y.; Cho, H.; Kim, S.; Seo, J.Y.; Cho, H.D.; Kang, H.C.; et al. Liposomal fusion of plant-based extracellular vesicles to enhance skin anti-inflammation. J. Ind. Eng. Chem. 2024. [Google Scholar] [CrossRef]
- Ko, H.J.; Sim, S.A.; Park, M.H.; Ryu, H.S.; Choi, W.Y.; Park, S.M.; Lee, J.N.; Hyun, C.G. Anti-Photoaging Effects of Upcycled Citrus junos Seed Anionic Peptides on Ultraviolet-Radiation-Induced Skin Aging in a Reconstructed Skin Model. Int. J. Mol. Sci. 2024, 25, 1711. [Google Scholar] [CrossRef] [PubMed]
- Cario-André, M.; Briganti, S.; Picardo, M.; Nikaido, O.; Gall, Y.; Ginestar, J.; Taïeb, A. Epidermal reconstructs: A new tool to study topical and systemic photoprotective molecules. J. Photochem. Photobiol. B Biol. 2002, 68, 79–87. [Google Scholar] [CrossRef] [PubMed]
- Seki, T.; Chung, C.-K.; Mikami, H.; Oshima, Y. Oshima. Deoxyribonucleic acid homology and taxonomy of the genus Bacillus. Int. J. Syst. Bacteriol. 1978, 28, 182–189. [Google Scholar] [CrossRef]
- Cai, M.; Han, Y.; Zheng, X.; Xue, B.; Zhang, X.; Mahmut, Z.; Wang, Y.; Dong, B.; Zhang, C.; Gao, D.; et al. Synthesis of Poly-γ-Glutamic Acid and Its Application in Biomedical Materials. Materials 2024, 17, 15. [Google Scholar] [CrossRef] [PubMed]
- Jungersted, J.M.; Hellgren, L.I.; Jemec, G.B.; Agner, T. Lipids and skin barrier function—A clinical perspective. Contact Dermat. 2008, 58, 255–262. [Google Scholar] [CrossRef]
- Sakai, S.; Sayo, T.; Kodama, S.; Inoue, S. N-Methyl-L-serine stimulates hyaluronan production in human skin fibroblasts. Skin. Pharmacol. Appl. Skin. Physiol. 1999, 12, 276–283. [Google Scholar] [CrossRef] [PubMed]
- Yu, H.Y.; Yang, I.-J.; Lincha, V.R.; Park, I.S.; Lee, D.-U.; Shin, H.M. The Effects of the Fruits of Foeniculum vulgare on Skin Barrier Function and Hyaluronic Acid Production in HaCaT Keratinocytes. J. Life Sci. 2015, 5, 880–888. [Google Scholar] [CrossRef]
- Tricarico, P.M.; Mentino, D.; De Marco, A.; Del Vecchio, C.; Garra, S.; Cazzato, G.; Foti, C.; Crovella, S.; Calamita, G. Aquaporins Are One of the Critical Factors in the Disruption of the Skin Barrier in Inflammatory Skin Diseases. Int. J. Mol. Sci. 2022, 23, 4020. [Google Scholar] [CrossRef] [PubMed]
- Screaton, G.R.; Bell, M.V.; Jackson, D.G.; Cornelis, F.B.; Gerth, U.; Bell, J.I. Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons. Proc. Natl. Acad. Sci. USA 1992, 89, 12160–12164. [Google Scholar] [CrossRef] [PubMed]
- Aruffo, A.; Stamenkovic, I.; Melnick, M.; Underhill, C.B.; Seed, B. CD44 is the principal cell surface receptor for hyaluronate. Cell 1990, 61, 1303–1313. [Google Scholar] [CrossRef]
- Li, J.; Tang, H.; Hu, X.; Chen, M.; Xie, H. Aquaporin-3 gene and protein expression in sun-protected human skin decreases with skin ageing. Australas. J. Dermatol. 2010, 51, 106–112. [Google Scholar] [CrossRef]
- Rockstroh, T. Changes in the nomenclature of bacteria after the 8th edition of Bergey’s Manual of the Determinative Bacteriology. Z. Fur Arztl. Fortbild. 1977, 71, 545–550. [Google Scholar]
- Birrer, G.A.; Cromwick, A.M.; Gross, R.A. Gamma-poly(glutamic acid) formation by Bacillus licheniformis 9945a: Physiological and biochemical studies. Int. J. Biol. Macromol. 1994, 16, 265–275. [Google Scholar] [CrossRef]
- Chettri, R.; Bhutia, M.O.; Tamang, J.P. Poly-γ-Glutamic Acid (PGA)-Producing Bacillus Species Isolated from Kinema, Indian Fermented Soybean Food. Front. Microbiol. 2016, 7, 971. [Google Scholar] [CrossRef]
- Kang, S.E.; Rhee, J.H.; Park, C.; Sung, M.H.; Lee, I.H. Distribution of poly-γ-glutamate (γ-PGA) producers in Korean fermented foods, Cheongkukjang, Doenjang, and Kochujang. Food Sci. Biotechnol. 2005, 14, 704. [Google Scholar]
- Tian, G.; Fu, J.; Wei, X.; Ji, Z.; Ma, X.; Qi, G.; Chen, S. Enhanced expression of pgdS gene for high production of poly-gamma-glutamic aicd with lower molecular weight in Bacillus licheniformis WX-02. J. Chem. Technol. Biotechnol. 2014, 89, 1825–1832. [Google Scholar] [CrossRef]
- Goto, A.; Kunioka, M. Biosynthesis and Hydrolysis of Poly(γ-glutamic acid) from Bacillus subtilis IF03335. Biosci. Biotechnol. Biochem. 1992, 56, 1031–1035. [Google Scholar] [CrossRef] [PubMed]
- Ko, H.J.; Kim, J.; Ahn, M.; Kim, J.H.; Lee, G.S.; Shin, T. Ergothioneine alleviates senescence of fibroblasts induced by UVB damage of keratinocytes via activation of the Nrf2/HO-1 pathway and HSP70 in keratinocytes. Exp. Cell Res. 2021, 400, 112516. [Google Scholar] [CrossRef] [PubMed]
- Choi, J.W.; Lee, J.; Park, Y.I. 7,8-Dihydroxyflavone attenuates TNF-α-induced skin aging in Hs68 human dermal fibroblast cells via down-regulation of the MAPKs/Akt signaling pathways. Biomed. Pharmacother. 2017, 95, 1580–1587. [Google Scholar] [CrossRef] [PubMed]
- Han, D.; Kim, H.Y.; Lee, H.J.; Shim, I.; Hahm, D.H. Wound healing activity of gamma-aminobutyric Acid (GABA) in rats. J. Microbiol. Biotechnol. 2007, 17, 1661–1669. [Google Scholar]
Gene | Primer | Sequence (5′ to 3′) |
---|---|---|
FLG | Sense | AAGCTTCATGGTGATGCGAC |
Antisense | TCAAGCAGAAGAGGAAGGCA | |
IVL | Sense | ACCTAGCGGACCCGAAATAA |
Antisense | TGGAACAGCAGGAAAAGCAC | |
LOR | Sense | CACTGGGGTTGGGAGGTAGT |
Antisense | GCTCTCATGATGCTACCCGA | |
SPT | Sense | CTGCTGAAGTCCTCAAGGAGTA |
Antisense | GGTTCAGCTCATCACTCAGAATC | |
HMG-CoA | Sense | GATCCAGGAGCGAACCAA |
Antisense | GCGAATAGACACACCACGTT | |
FAS | Sense | CCTCACTGCCATCCAGATTG |
Antisense | CTGTTTACATTCCTCCCAGGAC | |
HAS-1 | Sense | CCACCCAGTACAGCGTCAAC |
Antisense | CATGGTGCTTCTGTCGCTCT | |
HAS-2 | Sense | TTTGTTCAAGTCCCAGCAGC |
Antisense | ATCCTCCTGGGTGGTGTGAT | |
HAS-3 | Sense | CCCAGCCAGATTTGTTGATG |
Antisense | AGTGGTCACGGGTTTCTTCC | |
GAPDH | Sense | CAAAGTTGTCATGGATGACC |
Antisense | CCATGGAGAAGGCTGGGG |
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Ko, H.-J.; Park, S.; Shin, E.; Kim, J.; Lee, G.S.; Lee, Y.-J.; Park, S.M.; Lee, J.; Hyun, C.-G. Poly-γ-Glutamic Acid from a Novel Bacillus subtilis Strain: Strengthening the Skin Barrier and Improving Moisture Retention in Keratinocytes and a Reconstructed Skin Model. Int. J. Mol. Sci. 2025, 26, 983. https://doi.org/10.3390/ijms26030983
Ko H-J, Park S, Shin E, Kim J, Lee GS, Lee Y-J, Park SM, Lee J, Hyun C-G. Poly-γ-Glutamic Acid from a Novel Bacillus subtilis Strain: Strengthening the Skin Barrier and Improving Moisture Retention in Keratinocytes and a Reconstructed Skin Model. International Journal of Molecular Sciences. 2025; 26(3):983. https://doi.org/10.3390/ijms26030983
Chicago/Turabian StyleKo, Hyun-Ju, SeoA Park, Eunjin Shin, Jinhwa Kim, Geun Soo Lee, Ye-Jin Lee, Sung Min Park, Jungno Lee, and Chang-Gu Hyun. 2025. "Poly-γ-Glutamic Acid from a Novel Bacillus subtilis Strain: Strengthening the Skin Barrier and Improving Moisture Retention in Keratinocytes and a Reconstructed Skin Model" International Journal of Molecular Sciences 26, no. 3: 983. https://doi.org/10.3390/ijms26030983
APA StyleKo, H.-J., Park, S., Shin, E., Kim, J., Lee, G. S., Lee, Y.-J., Park, S. M., Lee, J., & Hyun, C.-G. (2025). Poly-γ-Glutamic Acid from a Novel Bacillus subtilis Strain: Strengthening the Skin Barrier and Improving Moisture Retention in Keratinocytes and a Reconstructed Skin Model. International Journal of Molecular Sciences, 26(3), 983. https://doi.org/10.3390/ijms26030983