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• 2017年03月   筑波大学   博士（農学）

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  日本細菌学会

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  日本微生物生態学会

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  日本農芸化学会

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  日本ウイルス学会

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• ライフサイエンス   ウイルス学  

• ライフサイエンス   応用微生物学  

• ライフサイエンス   細菌学  

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• 札幌医科大学   医学部 微生物学講座   助教  

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• RSウイルス

• ファージ

• 廃水処理

• バイオフィルム

• 脱窒

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• Bacillaceae serine proteases and Streptomyces epsilon-poly-l-lysine synergistically inactivate Caliciviridae by inhibiting RNA genome release

  Soh Yamamoto, Noriko Ogasawara, Yuka Sudo-Yokoyama, Sachiko Sato, Nozomu Takata, Nana Yokota, Tomomi Nakano, Kyoko Hayashi, Akira Takasawa, Mayumi Endo, Masako Hinatsu, Keitaro Yoshida, Toyotaka Sato, Satoshi Takahashi, Kenichi Takano, Takashi Kojima, Jun Hiraki, Shin-ich Yokota

  Scientific Reports ( Springer Science and Business Media LLC )  14 ( 1 )  2024年07月  [査読有り]

  　概要を見る

  Abstract Human norovirus (HuNoV) is an enteric infectious pathogen belonging to the Caliciviridae family that causes occasional epidemics. Circulating alcohol-tolerant viral particles that are readily transmitted via food-borne routes significantly contribute to the global burden of HuNoV-induced gastroenteritis. Moreover, contact with enzymes secreted by other microorganisms in the environment can impact the infectivity of viruses. Hence, understanding the circulation dynamics of Caliciviridae is critical to mitigating epidemics. Accordingly, in this study, we screened whether environmentally abundant secretase components, particularly proteases, affect Caliciviridae infectivity. Results showed that combining Bacillaceae serine proteases with epsilon-poly-l-lysine (EPL) produced by Streptomyces—a natural antimicrobial—elicited anti-Caliciviridae properties, including against the epidemic HuNoV GII.4_Sydney_2012 strain. In vitro and in vivo biochemical and virological analyses revealed that EPL has two unique synergistic viral inactivation functions. First, it maintains an optimal pH to promote viral surface conformational changes to the protease-sensitive structure. Subsequently, it inhibits viral RNA genome release via partial protease digestion at the P2 and S domains in the VP1 capsid. This study provides new insights regarding the high-dimensional environmental interactions between bacteria and Caliciviridae, while promoting the development of protease-based anti-viral disinfectants.

  DOI

• Disruption of SMC-related genes promotes recombinant cholesterol esterase production in Burkholderia stabilis

  Kenji Konishi, Yoshiaki Yasutake, Shuji Muramatsu, Satomi Murata, Keitaro Yoshida, Koji Ishiya, Sachiyo Aburatani, Shin-ichi Sakasegawa, Tomohiro Tamura

  Applied Microbiology and Biotechnology ( Springer Science and Business Media LLC )  106 ( 24 ) 8093 - 8110  2022年12月

  DOI

• Bacterial triacylglycerol lipase is a potential cholesterol esterase: Identification of a key determinant for sterol-binding specificity

  Yoshiaki Yasutake, Kenji Konishi, Shuji Muramatsu, Keitaro Yoshida, Sachiyo Aburatani, Shin-ichi Sakasegawa, Tomohiro Tamura

  International Journal of Biological Macromolecules ( Elsevier BV )  167   578 - 586  2021年01月  [査読有り]  [国際誌]

  　概要を見る

  Cholesterol esterase (Che) from Burkholderia stabilis (BsChe) is a homolog of well-characterized and industrially relevant bacterial triacylglycerol lipases (Lips). BsChe is a rare bacterial Lip enzyme that exhibits practical Che activity and is currently used in clinical applications to determine total serum cholesterol levels. To investigate the sterol specificity of BsChe, we determined the X-ray structure of BsChe. We discovered a local structural change in the active-site cleft, which might be related to substrate binding and product release. We also performed molecular docking studies by using the X-ray models of BsChe and cholesterol linoleate (CLL), the most favorable substrate for BsChe. The results showed that the sterol moieties of reasonable CLL docking poses localized to a specific active-site cleft surface formed by Leu266 and Ile287, which are unconserved among Burkholderia Lip homologs. Site-directed mutagenesis identified these residues as essential for the Che activity of BsChe, and Leu or Ile substitution conferred marked Che activity to Burkholderia Lips. In particular, Burkholderia cepacia and Burkholderia ubonensis Lips with the V266L/L287I double mutation exhibited ~50-fold and 500-fold higher Che activities than those of the wild-type enzymes, respectively. These results provide new insights into the substrate-binding mechanisms and selectivities of bacterial Lips.

  DOI PubMed

• Peculiarities of biofilm formation by Paracoccus denitrificans.

  Kana Morinaga, Keitaro Yoshida, Kohei Takahashi, Nobuhiko Nomura, Masanori Toyofuku

  Applied microbiology and biotechnology    2020年01月  [査読有り]  [国際誌]

  　概要を見る

  Most bacteria form biofilms, which are thick multicellular communities covered in extracellular matrix. Biofilms can become thick enough to be even observed by the naked eye, and biofilm formation is a tightly regulated process. Paracoccus denitrificans is a non-motile, Gram-negative bacterium that forms a very thin, unique biofilm. A key factor in the biofilm formed by this bacterium is a large surface protein named biofilm-associated protein A (BapA), which was recently reported to be regulated by cyclic diguanosine monophosphate (cyclic-di-GMP or c-di-GMP). Cyclic-di-GMP is a major second messenger involved in biofilm formation in many bacteria. Though cyclic-di-GMP is generally reported as a positive regulatory factor in biofilm formation, it represses biofilm formation in P. denitrificans. Furthermore, quorum sensing (QS) represses biofilm formation in this bacterium, which is also reported as a positive regulator of biofilm formation in most bacteria. The QS signal used in P. denitrificans is hydrophobic and is delivered through membrane vesicles. Studies on QS show that P. denitrificans can potentially form a thick biofilm but maintains a thin biofilm under normal growth conditions. In this review, we discuss the peculiarities of biofilm formation by P. denitrificans with the aim of deepening the overall understanding of bacterial biofilm formation and functions.

  DOI PubMed

• Production of recombinant extracellular cholesterol esterase using consistently active promoters in Burkholderia stabilis

  Keitaro Yoshida, Kenji Konishi, Arturo Magana-Mora, Adrien Rougny, Yoshiaki Yasutake, Shuji Muramatsu, Satomi Murata, Toshitaka Kumagai, Sachiyo Aburatani, Shin-ichi Sakasegawa, Tomohiro Tamura

  Bioscience, Biotechnology, and Biochemistry ( Oxford University Press (OUP) )  83 ( 10 ) 1974 - 1984  2019年10月  [査読有り]  [国際誌]

  担当区分：   筆頭著者

  　概要を見る

  Burkholderia stabilis FERMP-21014 produces highly active cholesterol esterase in the presence of fatty acids. To develop an overexpression system for cholesterol esterase production, we carried out RNA sequencing analyses to screen strongly active promoters in FERMP-21014. Based on gene expression consistency analysis, we selected nine genes that were consistently expressed at high levels, following which we constructed expression vectors using their promoter sequences and achieved overproduction of extracellular cholesterol esterase under fatty acid-free conditions. Of the tested promoters, the promoter of BSFP_0720, which encodes the alkyl hydroperoxide reductase subunit AhpC, resulted in the highest cholesterol esterase activity (24.3 U mL−1). This activity level was 243-fold higher than that of the wild-type strain under fatty acid-free conditions. We confirmed that cholesterol esterase was secreted without excessive accumulation within the cells. The gene expression consistency analysis will be useful to screen promoters applicable to the overexpression of other industrially important enzymes.

  DOI PubMed

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• 多剤耐性細菌感染症の切り札 ファージセラピー

  吉田圭太朗, 安藤弘樹

  臨床化学   51 ( 1 ) 47 - 47  2022年01月

  担当区分：   筆頭著者

• Burkholderia stabilis由来コレステロールエステラーゼの結晶構造

  安武義晃, 安武義晃, 小西健司, 小西健司, 村松周治, 吉田圭太朗, 油谷幸代, 油谷幸代, 油谷幸代, 酒瀬川信一, 田村具博, 田村具博, 田村具博

  量子ビームサイエンスフェスタ(Web)   2020  2021年

  J-GLOBAL

• Burkholderia stabilisを宿主とした細胞外コレステロールエステラーゼの組換え生産

  吉田圭太朗, 小西健司, MAGANA-MORA Arturo, MAGANA-MORA Arturo, ROUGNY Adrien, ROUGNY Adrien, 安武義晃, 安武義晃, 村松周治, 村田里美, 熊谷俊高, 油谷幸代, 油谷幸代, 酒瀬川信一, 田村具博, 田村具博, 田村具博

  日本農芸化学会大会講演要旨集(Web)   2020  2020年

  J-GLOBAL

• Burkholderia stabilisのコレステロールエステラーゼはT2SS機構の内膜タンパク質GspFを介して分泌される

  吉田圭太朗, 小西健司, 村松周治, 酒瀬川信一, 安武義晃, 油谷幸代, 田村具博

  日本農芸化学会西日本支部大会およびシンポジウム講演要旨集   2019  2019年

  J-GLOBAL

• Burkholderia stabilis由来コレステロールエステラーゼの機能と臨床検査薬への適用

  小西健司, 吉田圭太朗, 太田喜之, 安武義晃, 村松周治, 村田里美, 油谷幸代, 酒瀬川信一, 田村具博

  日本農芸化学会西日本支部大会およびシンポジウム講演要旨集   2019  2019年

  J-GLOBAL

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• 複合微生物系における膜の目詰まりの新奇生物学的制御

  研究期間:

  2014年04月

  -

  2017年03月

   

  吉田圭太朗

  担当区分： 研究代表者

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• Recombinant production of extracellular cholesterol esterase using consistently active promoters in Burkholderia stabilis

  Keitaro Yoshida

  2019 International Conference on Biotechnology and Bioengineering  

  発表年月： 2019年09月