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β-defensin 2 synthesized by a cell-free protein synthesis system and encapsulated in liposomes inhibits adhesion of Porphyromonas gingivalis to oral epithelial cells

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Abstract

β-defensin 2 (BD-2), an antimicrobial peptide (AMP), is expressed by oral epithelial cells and plays an important role in innate immunity of the oral cavity. Cell-free protein synthesis (CFPS) systems have been studied for the synthesis of various proteins, however, the synthesis of BD-2 by a CFPS system has not been extensively explored. Liposomes have been developed as tools for drug delivery. A delivery of liposome-encapsulated AMP to oral epithelium may be useful to prevent oral infectious diseases. In the present study, we investigated the antimicrobial activity of the BD-2 protein, artificially synthesized using a CFPS system and encapsulated in liposomes. BD-2 protein was artificially synthesized using template DNA and a reconstituted CFPS system and was identified by western blotting. Bilayer liposomes were prepared using 1,2-dioleoyl-sn-glycero-3-phospho-choline and 3-sn-phosphatidylcholine from egg yolk. The artificially synthesized BD-2 was encapsulated in liposomes, collected by ultrafiltration, and detected by western blotting. Human oral epithelial cells were cultured with the liposome-encapsulated BD-2 and the concentration of BD-2 in the cell lysate of the culture with the synthesized BD-2 was higher than that of the control cultures. The antimicrobial activity of the synthesized BD-2 was investigated by an adhesion assay of Porphyromonas gingivalis to oral epithelial cells. The artificially synthesized BD-2 and its liposome significantly inhibited adhesion of P. gingivalis to oral epithelial cells. These results suggest that artificially synthesized BD-2 and liposome-encapsulated BD-2 show antimicrobial activity and can potentially play a role in oral healthcare for periodontal diseases.

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Data Availability

All data supporting the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

This study was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (17H04418, 20K09941, 20K23083 and 22K17040). We thank GeneFrontier Co., Ltd. (Chiba, Japan) for supporting the design of the template DNA sequences with AT-rich codons for BD-2 synthesis.

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Authors and Affiliations

  1. Department of Oral Microbiology, Tokushima University, 3-18-15, Kuramoto, Tokushima, 770-8504, Japan

    Yuka Hiroshima

  2. Department of Periodontology and Endodontology, Tokushima University, Tokushima, Japan

    Jun-ichi Kido, Rie Kido, Mika Bando & Hiromichi Yumoto

  3. Department of Oral Healthcare Education, Graduate School of Biomedical Sciences, Tokushima University, Tokushima, Japan

    Kaya Yoshida

  4. Health and Medical Research Institute, National Institute of Advanced Industrial Science and Technology, Tokyo, Japan

    Kazuaki Kajimoto

  5. Institute of Advanced Medical Sciences, Tokushima University, Tokushima, Japan

    Yasuo Shinohara

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  1. Yuka Hiroshima
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Contributions

YH: Planning study, performing experiments (template DNA preparation, synthesized BD-2 analysis, bacterial adhesion assay etc.), writing and reviewing a manuscript and summarizing the study. JK: Co-planning study, performing experiments (liposome preparation, western blotting and ELISA), and supporting the preparation of figures in the manuscript. RK: Performing experiments (cell culture, ELISA and western blotting). KY: Observing liposomes. MB: Supporting SDS-PAGE and western blotting procedures, KK: Instructing liposome preparation and delivery experiments. HY: Advising study performance. YS: Instructing cell-free protein synthesis and evaluating the result data.

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Correspondence to Yuka Hiroshima.

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Hiroshima, Y., Kido, Ji., Kido, R. et al. β-defensin 2 synthesized by a cell-free protein synthesis system and encapsulated in liposomes inhibits adhesion of Porphyromonas gingivalis to oral epithelial cells. Odontology 111, 830–838 (2023). https://doi.org/10.1007/s10266-023-00789-x

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