2021 Fiscal Year Research-status 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 (EHEC) is one of the pathogenic E. coli capable of producing Shiga-like toxins (Stx). Antibiotics are not recommended as treatment for patients infected with EHEC strains because it will induce the release of Stx causing serious human gastrointestinal disease. Thus, there is no good treatment options as for now. This study aims to development a new therapeutic agent, termed Cas13a-stx, for treatment of EHEC infections by packaging CRISPR-Cas13a designed to target stx into E. coli phage capsid. Cas13a-stx can infect EHEC and sequence-specifically recognize stx gene, then promiscuously cleave surrounding ssRNA molecules, leading to decrement of toxin production and bacterial death.
To achieve the research aims, the following experiments are underway: 1) bacteriophages have been isolated from various sources and their lytic activities are checked against multiple species of E. coli including EHEC strains and clinical strains. 2) The Cas13a targeting stx is designed and 3) loaded into the capsid of the isolated phage candidate. 4) The effect of Cas13a-stx in protecting cell damage from Stx action is verified in vitro using Vero cells and 5) in vivo using mice model.
Currently, 5 EHEC strains have already been confirmed to carry stx gene and the candidate phage that can infect EHEC strains is successfully isolated from sewage water. Next, whole genome sequence in this phage will be determined before it was used to package Cas13a targeting stx.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
In the R3 research year, five stx-positive EHEC strains have already been identified. The design of Cas13a targeting stx which specifically recognize and kill EHEC strains and isolation of phage with satisfactory infectivity against the EHEC strains (3/5) are completed as planned.
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| Strategy for Future Research Activity |
The genome sequence of the candidate phage isolated from sewage water in Jichi Medical University will be determined and virulence-associated genes such as stx gene and endolysin will be deleted. Next, morphological characterization of the candidate phage will be performed using transmission electron microscopy (TEM) and the characterized phage will be loaded with the designed Cas13a targeting stx. Finally, cytotoxic effect and sequence-specific killing of Cas13a-stx will be evaluated using mammalian cell line (Vero cell) and mouse model, respectively.
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