Metal-Binding Antimicrobial Peptide SIF₄ Kills Escherichia coli by Targeting Cytoplasmic Biomacromolecules without Cytoplasmic Membrane Damage: A Mechanistic Study

To explore how metal-binding antimicrobial peptide SIF₄ kills foodborne Escherichia coli by targeting nucleic acid and protein in the cytoplasmic membrane without cytoplasmic membrane damage, the effect of SIF₄ on intracellular nucleic acid biosynthesis was investigated, and fluorescence spectral analysis of the competition between SIF₄ and ethidium bromide (EB) for binding to genomic DNA, ultraviolet (UV) spectral analysis of the interaction between SIF₄ and genomic DNA, and the binding mode between SIF₄ and genomic DNA were studied. Besides, the effect of SIF₄ on intracellular protein biosynthesis was systematically evaluated. Results demonstrated that SIF₄ could bind to E. coli genomic DNA through groove insertion and inhibit dose-dependently nucleic acid biosynthesis. Fluorescence spectral analysis showed that SIF₄ could compete with EB for binding to genomic DNA through intercalation binding and electrostatic adsorption. UV spectroscopy showed that combination with SIF₄ changed the molecular conformation of genomic DNA, but did not break its double strand structure. Circular dichroism (CD) spectroscopy showed that the base stacking force of genomic DNA was weakened, the double helix structure became loose, and the genomic DNA structure was changed from B to C configuration after combination with SIF₄. In addition, SIF₄ could significantly affect intracellular protein biosynthesis, and its inhibition effect was positively correlated with the treatment time and dose of SIF₄. It is believed that SIF₄ can enter the DNA groove through electrostatic adsorption or intercalation with genomic DNA, affecting DNA replication, RNA transcriptional biomass and protein translation to produce antimicrobial effect against Escherichia coli without cytoplasmic membrane damage. These results can provide support for the biocontrol of foodborne E. coli.