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

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

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

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

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