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. 2023 Mar 28;33(3):329-338.
doi: 10.4014/jmb.2207.07065. Epub 2023 Jan 28.

Screening of Anti-Adhesion Agents for Pathogenic Escherichia coli O157:H7 by Targeting the GrlA Activator

Sin Young Hong  1 , Byoung Sik Kim  1
Affiliations

Screening of Anti-Adhesion Agents for Pathogenic Escherichia coli O157:H7 by Targeting the GrlA Activator

Sin Young Hong et al. J Microbiol Biotechnol. .

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: Antibiotic alternatives; EHEC; Escherichia coli O157:H7; LEE operon; high-throughput screening (HTS); host cell adhesion.

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Conflict of interest statement

Conflict of Interest

S.Y.H. and B.S.K. have submitted a patent application covering this research. The authors have no financial conflicts of interest to declare.

Figures

Fig. 1
Fig. 1. Validation of GrlA as a target for an anti-adhesion agent.
(A) Schematic diagram of the GrlA-mediated regulation of LEE operons. The LEE operons (LEE1 to LEE5) and a bicistronic grlRA operon located between LEE1 and LEE2 are shown. When EHEC detects mechanical stimuli in the host intestine during initial attachment, membrane-bound inactive GrlA is released from the membrane and moves to the cytoplasm where it can activate the transcription of ler by binding to the LEE1 promoter. The expressed Ler then activates LEE operons as well as grlRA. GrlR interacts with the helix-turn-helix motif of GrlA and inhibits it. (B) Growth curve of E. coli EDL933 WT and ΔgrlA. The strains were cultured in LB medium at 37°C, and the A600 was measured hourly. (C) HeLa cells were cultured on a 24-well cell culture plate and co-incubated with the indicated EDL933 strains at an MOI of 100. After 2 h, the bacteria bound to the HeLa cells were quantified and expressed as the number of bacteria per well. The data were represented as the means ± standard deviation (SD) from three independent experiments. The Student’s t-test was used to determine the statistical significance (*, p < 0.05).
Fig. 2
Fig. 2. Reporter strain for the HTS of the GrlA inhibitor.
(A) A heterologous host E. coli DH5α reporter strain contains pSY2101 expressing GrlA under the PBAD promoter and pSY2104 carrying luxCDABE genes under the GrlA-activated promoter PLEE1. (B) The constructed reporter strain was verified by L-(+) arabinose induction, with various concentrations (w/ v) indicated. The one-way ANOVA with multiple comparison was used to determine the statistical significance between the control (no arabinose) and other samples at 4h (ns, non-significant; ****, p < 0.0001).
Fig. 3
Fig. 3. Validation of the initial hit molecules from the HTS.
(A) Validation using the heterologous host reporter strain SY2102. The strain was grown in LB medium supplemented with 0.0001% L-(+) arabinose and 2% DMSO or each chemical (final concentration of 20 μM). As a positive control, SY2102 was grown without L-(+) arabinose. (B) Validation using the homologous host reporter strain SY2110. The strain was grown in LB medium supplemented with 2% DMSO or each chemical (20 μM). As a positive control, SY2111 was grown in LB medium supplemented with 2% DMSO. After 5 h of incubation, the luminescence and absorbance were measured to calculate the % inhibition. The data were represented as the means ± SD from three independent experiments. The red arrows indicate the selected six compounds at this validation step with the homologous host reporter strain SY2110.
Fig. 4
Fig. 4. Validation of the hit molecules using strains expressing grlA*.
An additional set of heterologous (SY2201) and homologous (SY2204) host reporter strains expressing grlA* were prepared and used for the hit validation. (A) Strain SY2201 was grown in LB medium supplemented with 0.0002% L-(+) arabinose and the indicated hit molecules (20 μM). The negative and positive controls were the same strain grown with or without L-(+) arabinose, respectively. (B) Strain SY2204 was grown in LB with the indicated hit molecules (20 μM). Strains SY2204 and SY2111 treated with 2% DMSO were used as a negative and positive control, respectively. The RLU of each sample was calculated after 4 h of incubation. The data were represented as the means ± SD from three independent experiments. The red arrows indicate the selected two compounds at these validation steps. The Student’s t-test was used to determine the statistical significance between the negative control and other samples (ns, non-significant; ****, p < 0.0001; ***, p < 0.001; **, p < 0.01).
Fig. 5
Fig. 5. GrlA inhibitor, Grlactin.
The relative expression levels of the ler (LEE1) and espZ (LEE2) genes from the indicated samples were determined by qRT-PCR and expressed using that from the DMSO-treated WT strain sample as 1. The data were represented as the means ± SD from three independent experiments. The Student’s t-test was used to determine the statistical significance between the DMSO-treated WT and other samples (ns, non-significant; ****, p < 0.0001; ***, p < 0.001; **, p < 0.01).
Fig. 6
Fig. 6. Effects of Grlactin on the expression of the LEE genes and the adhesion of EHEC to the host cells.
(A) Effects of Grlactin on the expression level of each LEE operon gene were examined by qRT-PCR. (B) Reduction in EHEC adhesion to the HeLa cells after Grlactin treatment. The data were represented as the means ± SD from three independent experiments. The Student’s t-test was used to determine the statistical significance between the DMSO-treated WT and other samples (***, p < 0.001; **, p < 0.01; *, p < 0.05).
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