Glycan-Adhering Lectins and Experimental Evaluation of a Lectin FimH Inhibitor in Enterohemorrhagic Escherichia coli (EHEC) O157:H7 Strain EDL933

Abstract

In this study, we tried to develop a FimH inhibitor that inhibits adhesion of enterohemorrhagic Escherichia coli (EHEC) on the epithelium of human intestine during the initial stage of infections. Using a T7 phage display method with a reference strain, EHEC EDL933, FimH was selected as an adherent lectin to GM1a and Gb3 glycans. In order to detect the ligand binding domain (LBD) of FimH, we used a docking simulation and found three binding site sequences of FimH, i.e., P1, P2, and P3. Among Gb3 mimic peptides, P2 was found to have the strongest binding strength. Moreover, in vitro treatment with peptide P2 inhibited binding activity in a concentration-dependent manner. Furthermore, we conducted confirmation experiments through several strains isolated from patients in Korea, EHEC NCCP15736, NCCP15737, and NCCP15739. In addition, we analyzed the evolutionary characteristics of the predicted FimH lectin-like adhesins to construct a lectin-glycan interaction (LGI). We selected 70 recently differentiated strains from the phylogenetic tree of 2240 strains with Shiga toxin in their genome. We can infer EHEC strains dynamically evolved but FimH was conserved during the evolution time according to the phylogenetic tree. Furthermore, FimH could be a reliable candidate of drug target in terms of evolution. We examined how pathogen lectins interact with host glycans early in infection in EDL933 as well as several field strains and confirmed that glycan-like peptides worked as an initial infection inhibitor.

Keywords: FimH; Gb3; enterohemorrhagic Escherichia coli (EHEC); glycan; lectin.

Figure 1

cDNA synthesis and the plaque lift assay revealed lectin factors linked to Gb3 and GM1a glycan in EDL933. (A) cDNA synthesis. (B,C) The lectin factors associated with Gb3 and GM1a glycans were identified using a plaque lift assay.

Figure 2

Docking simulations with FimH, a lectin candidate that binds to Gb3 and GM1a glycans, were used to determine binding affinity. (A) Binding affinity was determined using docking simulations with FimH. (B) The binding strength (kcal/mol) to FimH lectins was stronger for Gb3 than for GM1a, and three LBDs at the expected binding sites were discovered.

Figure 3

Elucidation of inhibitor restraining linkage of EDL933 strain. (A) A comparison of the binding affinities of Gb3-replica peptides to FimH in EDL933 was made. The term “control” refers to the binding affinity of a matrix without peptides. When compared to the control, the P1–P3 peptides revealed statistically significant changes. (B) In vitro treatment with peptide P2 inhibited binding activity in a concentration-dependent manner. EDL933 were treated with compounds at 0, 10, 100, and 1000 nM. When compared to the control, the P1–P3 peptides revealed statistically significant changes. * Means were substantially different (* p < 0.05 and ** p < 0.01).

Figure 4

Elucidation of inhibitor restraining linkage of pathogenic enteric bacteria EHEC stains (NCCP15736, NCCP15737, and NCCP15739). (A) A comparison of the binding affinities of Gb3-replica peptides to FimH in EHEC stains (NCCP15736, NCCP15737, and NCCP15739) was made. The term “control” refers to the binding affinity of a matrix without peptides. When compared to the control, the P1–P3 peptides revealed statistically significant changes. (B) In vitro treatment with peptide P2 inhibited binding activity in a concentration-dependent manner. NCCP15736, NCCP15737, and NCCP15739 were treated with compounds at 0, 10, 100, and 1000 nM. When compared to the control, the P2 peptides revealed statistically significant changes. * Means were substantially different (* p < 0.05 and ** p < 0.01).

Figure 5

Multi-locus sequence typing (MLST)-based phylogenetic tree of EHEC strains. MLST-based phylogeny and phylogenetic tree of EHEC strains with respect to the FimH gene. (A) Evolutionary time scaled by 100; lower values imply relatively recent branching. The scale indicates the number of substitutions per site. (B) Evolutionary time scaled by 100; lower values imply relatively recent branching. The scale indicates the number of substitutions per site. Green: reference stain, Red: the stains isolated from patients.

Figure 6

Diagrammatic illustration of elucidating an inhibitor-restraining connection in the EDL933 of pathogenic enteric bacteria. Immunotherapy for EHEC EDL933 might be used to prevent and cure infectious disorders by determining the involvement of the host Gb3 glycan peptide in the immune response against the pathogen.