LLB Phage: Novel approach to eliminate food poisoning bacteria

• Project/Area Number: 18K14378
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 38020:Applied microbiology-related
• Research Institution: Kyushu University
• Principal Investigator: Masuda Yoshimitsu 九州大学, 農学研究院, 助教 (90778060)
• Project Period (FY): 2018-04-01 – 2022-03-31
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000); Fiscal Year 2021: ¥650,000 (Direct Cost: ¥500,000、Indirect Cost: ¥150,000); Fiscal Year 2020: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000); Fiscal Year 2019: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000); Fiscal Year 2018: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
• Keywords: bacteriophage / bacteriocin / leaderless bacteriocin / CRISPR-Cas / EHEC / MRSA / Escherichia coli / Staphylococcus aureus / バクテリオファージ / リーダレスバクテリオシン / 食中毒細菌 / bacterial phage / food poisoning bacteria

Outline of Final Research Achievements

This study was designed to construct a novel antimicrobial agent, LLB-producing phage (LLB-phage) against pathogenic bacteria. As a result of this study, the first LLB-phage lnqQ-T7, LLB-ECP52s targeting enterohemorrhagic Escherichia coli, and lnqQ-vPSARa targeting multi-drug resistant Staphylococcus aureus were constructed.
lnqQ-T7 was constructed by trx-dependent homologous recombination system and LLB-ECP52s were constructed by genome editing system with CRISPR-Cas9. They could control the growth of not only E. coli host strain but neighboring gram-positive bacteria by enhanced lytic activity and LLB production. lnqQ-T7 could also inhibit an emergence of phage resistant population. MRSA targeting LLB phage lnqQ-vPSARa was constructed by editing system with CRISPR-Cas10, in which we could transform temperate phage PSARa into virulent phage by deleting 4 putative lysogenic genes.
This study demonstrated the great possibility that we can design various types of LLB-phages on demand.

Academic Significance and Societal Importance of the Research Achievements

本研究は、細菌にのみ感染し、薬剤耐性菌に対しても強い抗菌活性を有するバクテリオファージと抗菌ペプチドの一種であるリーダーレスバクテリオシンを遺伝的手法を駆使して組み合わせることで、LLBファージという新規抗菌素材を生み出し、食中毒細菌の制御等に利用することを目的とした。その結果、大腸菌O157H7株や多剤耐性黄色ブドウ球菌（MRSA）に対して非常に高い抗菌活性を有する優れたLLBファージを複数作成することに成功した。
また、今回構築したファージゲノム編集システムによって、ファージ利用で問題となる溶原化に関連する遺伝子を欠損させ、溶菌ファージ化させることにも成功した。

Research Products

• [Journal Article] Construction of Leaderless-Bacteriocin-Producing Bacteriophage Targeting E. coli and Neighboring Gram-Positive Pathogens (2021)
  • Author(s): Masuda Yoshimitsu、Kawabata Shun、Uedoi Tatsuya、Honjoh Ken-ichi、Miyamoto Takahisa
  • Journal Title: Microbiology Spectrum Volume: 9 Issue: 1
  • DOI: 10.1128/spectrum.00141-21
  • Peer Reviewed / Open Access
• [Presentation] Construction Of Leaderless-bacteriocin-producing Phage To Control Both Gram-negative And -positive Pathogenic Bacteria (2021)
  • Author(s): Yoshimitsu Masuda, Shun Kawabata, Tatsuya Uedoi, Ken-ichi Honjoh, Takahisa Miyamoto
  • Organizer: WORLD MICROBE FORUM
  • Int'l Joint Research
• [Presentation] リーダーレスバクテリオシン産生バクテリオファージの構築 (2021)
  • Author(s): 益田時光、川畑淳、上土井達哉、本城賢一、宮本敬久
  • Organizer: 九州微生物研究フォーラム
• [Presentation] 黄色ブドウ球菌を標的宿主とするaureocin A53 産生ファージの構築 (2021)
  • Author(s): 木下慧斗，井門さら，上土井達哉，益田時光，本城賢一，宮本敬久
  • Organizer: （公社）日本食品科学工学会第68回大会
• [Presentation] CRISPR -Cas10 システムによる，黄色ブドウ球菌を標的宿主としたLLB ファージの構築とその利用 (2021)
  • Author(s): 井門さら，木下慧斗，益田時光，本城賢一，宮本敬久
  • Organizer: （公社）日本食品科学工学会第68回大会
• [Presentation] 病原性大腸菌を標的宿主としたリーダーレスバクテリオシン産生ファージの構築とその利用に関する研究 (2021)
  • Author(s): 上土井達哉，川畑諄，益田時光，本城賢一，宮本敬久
  • Organizer: （公社）日本食品科学工学会第68回大会
• [Presentation] Construction of leaderless bacteriocin producing phage (2019)
  • Author(s): Yoshimitsu Masuda, Shun Kawabata, Hen-ichi Honjoh, Takahisa Miyamoto
  • Organizer: FEMS2019
  • Int'l Joint Research