Visualizing Attack of <i>Escherichia coli</i> by the Antimicrobial Peptide Human Defensin 5

Abstract

Human α-defensin 5 (HD5) is a 32-residue cysteine-rich host-defense peptide that exhibits broad-spectrum antimicrobial activity and contributes to innate immunity in the human gut and other organ systems. Despite many years of investigation, its antimicrobial mechanism of action remains unclear. In this work, we report that HD5_ox, the oxidized form of this peptide that exhibits three regiospecific disulfide bonds, causes distinct morphological changes to <i>Escherichia coli</i> and other Gram-negative microbes. These morphologies include bleb formation, cellular elongation, and clumping. The blebs are up to ∼1 μm wide and typically form at the site of cell division or cell poles. Studies with <i>E. coli</i> expressing cytoplasmic GFP reveal that HD5_ox treatment causes GFP emission to localize in the bleb. To probe the cellular uptake of HD5_ox and subsequent localization, we describe the design and characterization of a fluorophore–HD5 conjugate family. By employing these peptides, we demonstrate that fluorophore–HD5_ox conjugates harboring the rhodamine and coumarin fluorophores enter the <i>E. coli</i> cytoplasm. On the basis of the fluorescence profiles, each of these fluorophore–HD5_ox conjugates localizes to the site of cell division and cell poles. These studies support the notion that HD5_ox, at least in part, exerts its antibacterial activity against <i>E. coli</i> and other Gram-negative microbes in the cytoplasm