LLB Phage: Novel approach to eliminate food poisoning bacteria

2021 Fiscal Year Final Research Report

• 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
• Keywords: bacteriophage / bacteriocin / leaderless bacteriocin / CRISPR-Cas / EHEC / MRSA

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.

Free Research Field

分子微生物

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。