The Antimicrobial Activity of Gramicidin A Is Associated with Hydroxyl Radical Formation

<div><p>Gramicidin A is an antimicrobial peptide that destroys gram-positive bacteria. The bactericidal mechanism of antimicrobial peptides has been linked to membrane permeation and metabolism disruption as well as interruption of DNA and protein functions. However, the exact bacterial killing mechanism of gramicidin A is not clearly understood. In the present study, we examined the antimicrobial activity of gramicidin A on <i>Staphylococcus aureus</i> using biochemical and biophysical methods, including hydroxyl radical and NAD⁺/NADH cycling assays, atomic force microscopy, and Fourier transform infrared spectroscopy. Gramicidin A induced membrane permeabilization and changed the composition of the membrane. The morphology of <i>Staphylococcus aureus</i> during gramicidin A destruction was divided into four stages: pore formation, water permeability, bacterial flattening, and lysis. Changes in membrane composition included the destruction of membrane lipids, proteins, and carbohydrates. Most interestingly, we demonstrated that gramicidin A not only caused membrane permeabilization but also induced the formation of hydroxyl radicals, which are a possible end product of the transient depletion of NADH from the tricarboxylic acid cycle. The latter may be the main cause of complete <i>Staphylococcus aureus</i> killing. This new finding may provide insight into the underlying bactericidal mechanism of gA.</p></div

FT-IR spectra of the chemical change in <i>S. aureus</i> treated with gA.

<p>(A) FT-IR spectra of the chemical change in <i>S. aureus</i> treated with different concentrations of gA. In the spectrum, I, II, III, and IV represent the four characteristic IR regions for <i>S</i>. <i>aureus</i>. (B) FT-IR spectra in region I (C-H vibration of bacterial membrane fatty acids), (C) FT-IR spectra in regions II (protein or peptide amide I and II), III (vibrations of proteins, fatty acids, and phosphate-carrying compounds), and IV (stretching vibration of functional groups of polysaccharides such as C-O), and (D) FT-IR spectra in region II for protein amide I and amide II.</p

Hydroxyl radical formation following treatment with gA.

<p>(A) The hydroxyl radical formation following treatment with 5 μg/ml gA for 1, 2, and 3 hr. (B) The hydroxyl radical formation following treatment with 0, 1, and 20 μg/ml gA for 3 hr. (C) A histogram of hydroxyl radical formation produced by treatment with different concentrations of gA. The hydroxyl radical level following treatment with 60 mM H₂O₂ was used as a positive control for comparison.</p

Growth curves of <i>S. aureus</i> following treatment with gA.

<p>(A) The growth curve of <i>S. aureus</i> (A) without treatment with gA. (B-D) The growth curves of <i>S</i>. <i>aureus</i> in the lag (B), exponential (C), and stationary (D) phases following treatment with different concentrations of gA (0.025–10 μg/ml).</p

Atomic force microscopic images of <i>S. aureus</i> in the exponential phase following treatment with gA.

<p>(A) AFM images in the absence of gA treatment, showing typical round <i>S</i>. <i>aureus</i> cells with a smooth surface. (B-E) AFM images in the presence of treatment with 5 μg/ml gA. In (B), characteristic pores (red circle) and blebs (blue arrow) on the membrane surface of <i>S</i>. <i>aureus</i> were observed. In (C), flat-shaped <i>S</i>. <i>aureus</i> cells were observed, indicating that the bacterial membrane was destroyed by treatment with gA. (D) The bacterial membrane was further disrupted. In (E), bacteria were completely destroyed and lysed by treatment with gA.</p

The surface lipophilicity distribution for ligand binding with receptor.

<p>The complex structure is represented as ribbon structure with the N-loop of the ligand colored green, the N-terminus of receptor colored pink, and the EC-loops colored yellow. Blue color represents the hydrophilic part while green color represents hydrophobic part. Residues around the binding interface are labeled and shown as sticks; black font is for receptor, while red font is for ligand. (A): Monomeric CXCL-8 binding with CXCR1 at the initial time. Hydrophobic pocket of ligand CXCL-8 is also marked. (B): Monomeric CXCL-8 binding with CXCR1 at the final simulation time. (C): CXCL-8 binding with CXCR1_mut at the initial time. (D): CXCL-8 binding with CXCR1_mut after the 300 ns runs.</p

<i>In Silico</i> Analysis Reveals Sequential Interactions and Protein Conformational Changes during the Binding of Chemokine CXCL-8 to Its Receptor CXCR1

<div><p>Chemokine CXCL-8 plays a central role in human immune response by binding to and activate its cognate receptor CXCR1, a member of the G-protein coupled receptor (GPCR) family. The full-length structure of CXCR1 is modeled by combining the structures of previous NMR experiments with those from homology modeling. Molecular docking is performed to search favorable binding sites of monomeric and dimeric CXCL-8 with CXCR1 and a mutated form of it. The receptor-ligand complex is embedded into a lipid bilayer and used in multi ns molecular dynamics (MD) simulations. A multi-steps binding mode is proposed: (i) the N-loop of CXCL-8 initially binds to the N-terminal domain of receptor CXCR1 driven predominantly by electrostatic interactions; (ii) hydrophobic interactions allow the N-terminal Glu-Leu-Arg (ELR) motif of CXCL-8 to move closer to the extracellular loops of CXCR1; (iii) electrostatic interactions finally dominate the interaction between the N-terminal ELR motif of CXCL-8 and the EC-loops of CXCR1. Mutation of CXCR1 abrogates this mode of binding. The detailed binding process may help to facilitate the discovery of agonists and antagonists for rational drug design.</p></div

MM/PBSA binding free energy calculations for human and rat CD81s to HCV E2 protein.

<p>(A) For different HCV E2 sites (E2-site1, E2-site2, and E2-both sites) binding to human and rat CD81s, the binding free energies of human CD81 to HCV E2 were lower than those of rat CD81. HCV E2-site2 bound to human CD81 with the lowest binding free energy (H-E2-S2). (B) The detailed analysis of the components of binding free energies showed that the major difference for HCV E2-site2 binding to human and rat CD81s lies in the electrostatic interactions (H-E2-S2 and R-E2-S2). VDW dominates the binding of HCV E2-site1 to human CD81 (H-E2-S1). The figure represents the following. For H-E2-S1: the E2-site1 binding to human CD81; for H-E2-S2: the E2-site2 binding to human CD81; for H-E2-both: E2-both sites binding to human CD81; for R-E2-S1: the E2-site1 binding to rat CD81; and for R-E2-S2: the E2-site2 binding to rat CD81.</p

Comparison of surface charge and lipophilicity distributions between human and rat CD81 ectodomains.

<p>The structures of the resolved human CD81 (gray) and the homology-modeled rat CD81 (pink) shown in the ribbon were superposed. The major differences between the two CD81 structures are at the flexible loops from 173 to 186 a.a. (green: human CD81; red: rat CD81). (B) The surface charge distributions of the two CD81s show that the rat CD81 is more positively charged than human CD81 at the flexible loop region marked with a dashed line (blue: positive charge; red: negative charge). (C) The lipophilicity maps do not show much difference between the human and rat CD81s in the loop region (blue: hydrophilic; green: lipophilic).</p

The distances between the charged groups of ligand and receptor forming electrostatic interactions.

<p>(A) Some positively charged residues of the N-loop of CXCL-8 gradually approach to the negatively charged residues of the N-terminus of CXCR1 by electrostatic interactions (K3^CXCL-8-D194^CXCR1 (black), K11^CXCL-8-D14^CXCR1 (red), K15^CXCL-8-D13^CXCR1 (green)) during 300 ns MD simulations. (B) Other positively charged residues of CXCL-8 interact with the negatively charged residues of CXCR1 by electrostatic interactions (R47^CXCL-8-D14^CXCR1 (pink), K64^CXCL-8-E35^CXCR1 (blue), R60^CXCL-8-E275^CXCR1 (yellow)) during 300 ns MD simulations. Distances are the average values with the function of time for three replicates of the system monomeric CXCL-8 binding to CXCR1. Error bars of the curves are omitted for figure clarity.</p