Discovery and Characterization of a Disulfide-Locked C2-Symmetric Defensin Peptide

We report the discovery of HD5-CD, an unprecedented C2-symmetric β-barrel-like covalent dimer of the cysteine-rich host-defense peptide human defensin 5 (HD5). Dimerization results from intermonomer disulfide exchange between the canonical α-defensin CysII–CysIV (Cys5–Cys20) bonds located at the hydrophobic interface. This disulfide-locked dimeric assembly provides a new element of structural diversity for cysteine-rich peptides as well as increased protease resistance, broad-spectrum antimicrobial activity, and enhanced potency against the opportunistic human pathogen Acinetobacter baumannii

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

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

Discovery and Characterization of a Disulfide-Locked <i>C</i>₂‑Symmetric Defensin Peptide

We report the discovery of HD5-CD, an unprecedented <i>C</i>₂-symmetric β-barrel-like covalent dimer of the cysteine-rich host-defense peptide human defensin 5 (HD5). Dimerization results from intermonomer disulfide exchange between the canonical α-defensin Cys^II–Cys^IV (Cys⁵–Cys²⁰) bonds located at the hydrophobic interface. This disulfide-locked dimeric assembly provides a new element of structural diversity for cysteine-rich peptides as well as increased protease resistance, broad-spectrum antimicrobial activity, and enhanced potency against the opportunistic human pathogen Acinetobacter baumannii

Discovery and Characterization of a Disulfide-Locked C[subscript 2]-Symmetric Defensin Peptide

We report the discovery of HD5-CD, an unprecedented C[subscript 2]-symmetric β-barrel-like covalent dimer of the cysteine-rich host-defense peptide human defensin 5 (HD5). Dimerization results from intermonomer disulfide exchange between the canonical α-defensin Cys[superscript II]–Cys[superscript IV] (Cys[superscript 5]–Cys[superscript 20]) bonds located at the hydrophobic interface. This disulfide-locked dimeric assembly provides a new element of structural diversity for cysteine-rich peptides as well as increased protease resistance, broad-spectrum antimicrobial activity, and enhanced potency against the opportunistic human pathogen Acinetobacter baumannii.United States. National Institutes of Health (DP2OD007045)United States. National Institutes of Health (PO1 GM047467)United States. National Institutes of Health (F32GM103005)United States. National Institutes of Health (EB-002026)National Science Foundation (U.S.) (Grant NSF-007031

Visualizing Attack of Escherichia coli by the Antimicrobial Peptide Human Defensin 5

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[subscript ox], the oxidized form of this peptide that exhibits three regiospecific disulfide bonds, causes distinct morphological changes to Escherichia coli 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 E. coli expressing cytoplasmic GFP reveal that HD5[subscript ox] treatment causes GFP emission to localize in the bleb. To probe the cellular uptake of HD5[subscript 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[subscript ox] conjugates harboring the rhodamine and coumarin fluorophores enter the E. coli cytoplasm. On the basis of the fluorescence profiles, each of these fluorophore–HD5[subscript ox] conjugates localizes to the site of cell division and cell poles. These studies support the notion that HD5[subscript ox'], at least in part, exerts its antibacterial activity against E. coli and other Gram-negative microbes in the cytoplasm.United States. Army Research Office. Institute for Soldier Nanotechnologies (Contract W911NF-13-D-0001)National Science Foundation (U.S.) (Grant 007031)Massachusetts Institute of Technology (MIT UROP Program funds)Royal Thai Government (RTG) (Fellowship)Massachusetts Institute of Technology (2014 Richard R. Schrock summer graduate fellowship)National Institutes of Health (U.S.) (NIH Office of the Director, grant DP2OD007045