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Recombinant production and evaluation of an antibacterial l-amino acid oxidase derived from flounder Platichthys stellatus

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Abstract

Fish produce mucus substances as a defensive outer barrier against several bacterial infections. We have recently identified an antibacterial l-amino acid oxidase (psLAAO1) in the mucus layer of the flounder Platichthys stellate. In this study, the antibacterial protein psLAAO1 was expressed as a secretory bioactive recombinant protein in the methylotrophic yeast Pichia pastoris. The recombinant psLAAO1 inhibited the growth of bacteria to the same levels as native psLAAO1 present in mucus. In particular, Staphylococci and Yersinia were strongly suppressed, showing the highest growth retardation of the 21 species and strains tested. Moreover, Staphylococcus epidermidis was most sensitive to psLAAO1 with a minimum inhibitory concentration (MIC) of 0.078 μg/mL, whereas Escherichia coli was essentially resistant to psLAAO1 with a MIC of >10 μg/mL. Interestingly, psLAAO1-treated E. coli were found to upregulate the expression of the btuE gene, which encodes glutathione peroxidase (GPx). The biochemical function of GPx is to reduce free hydrogen peroxide and is induced under response to reactive oxygen species (ROS). Thus, E. coli confers resistance to the reduced free hydrogen peroxide produced by psLAAO1 by increasing GPx levels. Furthermore, the growth of Staphylococcus aureus was completely inhibited in the presence of recombinant psLAAO1. The morphology of psLAAO1-treated S. aureus showed cell surface damage, the formation of large aggregates and the cells showed severe deformations. Western blot analysis showed that psLAAO1 binds to the surface of S. aureus. Therefore, psLAAO1 binds to the surface of LAAO-sensitive S. aureus and directs peroxidative activity at the surface of the bacterial membrane.

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Acknowledgments

We are grateful to Dr. Mineo Senda and Dr. Yoshimitsu Ohtomo for advice and discussion of this work. We thank Yutaro Takabuchi and Reina Tabata for helpful assistance, and Yusei Tsushima for technical support of the scanning electron microscopy analysis. This work was supported in part by a Hirosaki University Grant for Exploratory Research by Young Scientists.

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Authors and Affiliations

  1. Department of Pathologic Analysis, Division of Medical Life Sciences, Graduate School of Health Sciences, Hirosaki University, 66-1 Hon-cho, Hirosaki, Aomori, Japan

    Kosuke Kasai & Tomisato Miura

  2. Department of Clinical Laboratory, Yokohama City University Medical Center, Kanagawa, Japan

    Kenro Hashiguchi

  3. I’ROM Co., Ltd, Tokyo, Japan

    Hiroki Takahashi

  4. Department of Clinical Laboratory, Hirosaki Municipal Hospital, Aomori, Japan

    Ayano Kasai

  5. Department of Medical Technology, School of Health Sciences, Hirosaki University, Aomori, Japan

    Sadanori Takeda

  6. Department of Biomedical Sciences, Division of Medical Life Sciences, Graduate School of Health Sciences, Hirosaki University, Aomori, Japan

    Manabu Nakano, Takashi Ishikawa & Toshiya Nakamura

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  1. Kosuke Kasai
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  2. Kenro Hashiguchi
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  9. Tomisato Miura
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Correspondence to Tomisato Miura.

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Kasai, K., Hashiguchi, K., Takahashi, H. et al. Recombinant production and evaluation of an antibacterial l-amino acid oxidase derived from flounder Platichthys stellatus . Appl Microbiol Biotechnol 99, 6693–6703 (2015). https://doi.org/10.1007/s00253-015-6428-1

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  • DOI: https://doi.org/10.1007/s00253-015-6428-1

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