Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Screening of Anti-Adhesion Agents for Pathogenic Escherichia coli O157:H7 by Targeting the GrlA Activator

  • Sin Young Hong (Department of Food Science and Biotechnology, Ewha Womans University) ;
  • Byoung Sik Kim (Department of Food Science and Biotechnology, Ewha Womans University)
  • Received : 2022.07.29
  • Accepted : 2022.09.30
  • Published : 2023.03.28
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Abstract

Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen that produces attaching and effacing lesions on the large intestine and causes hemorrhagic colitis. It is primarily transmitted through the consumption of contaminated meat or fresh produce. Similar to other bacterial pathogens, antibiotic resistance is of concern for EHEC. Furthermore, since the production of Shiga toxin by this pathogen is enhanced after antibiotic treatment, alternative agents that control EHEC are necessary. This study aimed to discover alternative treatments that target virulence factors and reduce EHEC toxicity. The locus of enterocyte effacement (LEE) is essential for EHEC attachment to host cells and virulence, and most of the LEE genes are positively regulated by the transcriptional regulator, Ler. GrlA protein, a transcriptional activator of ler, is thus a potential target for virulence inhibitors of EHEC. To identify the GrlA inhibitors, an in vivo high-throughput screening (HTS) system consisting of a GrlA-expressing plasmid and a reporter plasmid was constructed. Since the reporter luminescence gene was fused to the ler promoter, the bioluminescence would decrease if inhibitors affected the GrlA. By screening 8,201 compounds from the Korea Chemical Bank, we identified a novel GrlA inhibitor named Grlactin [3-[(2,4-dichlorophenoxy)methyl]-4-(3-methylbut-2-en-1-yl)-4,5-dihydro-1,2,4-oxadiazol-5-one], which suppresses the expression of LEE genes. Grlactin significantly diminished the adhesion of EHEC strain EDL933 to human epithelial cells without inhibiting bacterial growth. These findings suggest that the developed screening system was effective at identifying GrlA inhibitors, and Grlactin has potential for use as a novel anti-adhesion agent for EHEC while reducing the incidence of resistance.

Keywords

Acknowledgement

This work was supported by the National Research Foundation of Korea funded by the Ministry of Science and ICT (NRF-2020R1F1A1070168) and the Ministry of Food and Drug Safety in 2022 (20162MFDS142) to B.S.K. The chemical library used in this study was kindly provided by the Korea Chemical Bank (www.chembank.org) of Korea Research Institute of Chemical Technology. We thank Dr. B.T. Kim at Korea Research Institute of Chemical Technology for depositing the Grlactin into the chemical library. We also thank Dr. S.H. Choi at Seoul National University for sharing the E. coli EDL933 ΔgrlA strain and HeLa cells. pCas and pTargetF were gifts from Sheng Yang (Addgene plasmid #62225 and #62226).

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