2022 Fiscal Year Annual Research Report
Development of a novel antimicrobial capable of selectively killing Shiga toxin-producing Escherichia coli
| Project/Area Number |
21K15436
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| Research Institution | Jichi Medical University |
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Principal Investigator |
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| Project Period (FY) |
2021-04-01 – 2023-03-31
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| Keywords | EHEC / Shiga toxin / CRISPR-Cas13a / Bacteriophage |
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| Outline of Annual Research Achievements |
Enterohemorrhagic E. coli or EHEC is capable of releasing Shiga-like toxins (Stx). The use of antibiotics is not a recommended therapeutic option for people infected with EHEC strains because it would trigger the release of Stx, leading to serious human gastrointestinal disease. Hence, there are currently no effective therapeutics available. This research aimed to develop a new therapeutic agent to treat EHEC infections which can be accomplished by packaging stx-targeting CRISPR-Cas13a into the capsid of an E. coli phage.
In the R3 research year, five stx-positive EHEC strains were collected. The stx-targeting Cas13a has also been designed and loaded into the capsid of a model narrow-range phage 80, generating Cas13a-stx. The bactericidal activity of Cas13a-stx was then confirmed. Furthermore, a candidate phage isolated from sewage water which has a satisfactory infectivity against EHEC strains (3/5) was isolated. It carried a genome larger than 200 kbp. Since there are difficulties in manipulating phages with large genomes, 706 clinical E. coli strains were chemically induced to further isolate broad-host-range phages.
In the R4 research year, a candidate broad-host-range phage (killing activity of 33.67%) with small genome size (about 30 kbp) was isolated. Unfortunately, this phage is not able to infect EHEC strains (0/5). Although this work does not progress as proposed, the use of CRISPR-Cas13a-loaded capsids for specific killing of E. coli expressing target sequence was confirmed, thus justifying the potential of this system as an alternative therapeutic against EHEC.
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