Abstract
Enterohaemorrhagic Escherichia coli (EHEC) cause the most severe E. coli infection with such as hemorrhagic colitis and hemorrhagic uremic syndrome (HUS). Shiga toxin is the major virulence factor during EHEC infection, and the gene encoding toxin is located on λ-like prophage region integrated into the bacterial chromosome. The expression of toxin is generally coupled to the phage induction through SOS response triggered by DNA damage. In a previous study showed that AbpAB can block the propagation of bacteriophages including λ phage. Here, we challenged whether AbpAB inhibits the phage propagation in E. coli o157:H7 EDL933, which is a model for EHEC, to prevent the toxin production and elucidate the mechanism. When the EHEC cells expressing AbpAB was cultured with mitomycin C (MMC) and ciprofloxacin (CPFX) to induce the phage and shiga toxin production, the plaque number and toxin level were decreased significantly. The result indicated that AbpAB inhibited phage production from the lysogenic strain. Growth curve of the strain showed that AbpAB prevented lysis of the EHEC strain under MMC and CPFX treatment. A disc diffusion test showed that inhibition zone of AbpAB expressing cells by MMC was bigger and its reporter signal was lower compared those without the expression. This result suggested that SOS response was blocked by AbpAB. To further elucidate the target of AbpAB, LexA degradation assay was performed to know whether RecA would be the target of AbpAB. As the result, AbpAB did not block the LexA degradation, while recA mutant did. Therefore, AbpAB might not target on RecA. It has been known that some doubled mutants (∆dam ∆recA, ∆dam ∆recB, ∆dam ∆recC) shows lethality. Thus, we transformed AbpAB plasmid into ∆dam strain and found that expression of AbpAB in ∆dam strain can grow. It indicated that AbpAB does not target on RecA, RecB, and RecC. Besides, a triple mutants strain (∆rep ∆recD ∆recA) also shows lethality, while the double mutants of them are survival. We found that a double mutant (∆rep ∆recA) expressing AbpAB showed lethality, and showed similar phenotype to the triple mutant. Based on these results, we suggested that AbpAB may target on RecD.This study demonstrated that AbpAB inhibited phage production through blocking SOS response in E. coli, while DNA got damage by MMC or CPFX. But, further study is necessary to understand the molecular mechanism in detail. Together, the results of this research would be applicable to develop a treatment strategy for shiga toxin production in EHEC.
| Date of Award | 2021 |
|---|---|
| Original language | English |
| Supervisor | Masayuki Hashimoto (Supervisor) |