Antibacterial action of structurally diverse cationic peptides on gram-positive bacteria

Abstract

Antimicrobial cationic peptides are ubiquitous in nature and are thought to be a component of the first line of defense against infectious agents. It is widely believed that the killing mechanism of these peptides on bacteria involves an interaction with the cytoplasmic membrane. Cationic peptides from different structural classes were used in experiments with Staphylococcus aureus and other medically important gram-positive bacteria to gain insight into the mechanism of action. The membrane potential-sensitive fluorophore dipropylthiacarbocyanine was used to assess the interactions of selected antimicrobial peptides with the cytoplasmic membrane of S. aureus. Study of the kinetics of killing and membrane depolarization showed that, at early time points, membrane depolarization was incomplete, even when 90% or more of the bacteria had been killed. CP26, a 26-amino-acid alpha-helical peptide with a high MIC against S. aureus, still had the ability to permeabilize the membrane. Cytoplasmic-membrane permeabilization was a widespread ability and an action that may be necessary for reaching an intracellular target but in itself did not appear to be the killing mechanism. Transmission electron microscopy of S. aureus and Staphylococcus epidermidis treated with CP29 (a 26-amino-acid alpha-helical peptide), CP11CN (a 13-amino-acid, proline- and tryptophan-rich peptide), and Bac2A-NH(2) (a linearized version of the 12-amino-acid loop peptide bactenecin) showed variability in effects on bacterial structure. Mesosome-like structures were seen to develop in S. aureus, whereas cell wall effects and mesosomes were seen with S. epidermidis. Nuclear condensation and abherrent septation were occasionally seen in S. epidermidis. Our experiments indicated that these peptides vary in their mechanisms of action and that the mechanism of action likely does not solely involve cytoplasmic-membrane permeabilization.

FIG. 1

Numbers of survivors (in log units) of S. aureus (A) and S. epidermidis (B) in the presence of CP11CN (+), CP29 (○), Bac2A-NH₂ (▵) and numbers of survivors of S. aureus (A) in the presence of indolicin (▿) and CP10A (□). All peptides were at concentrations 10 times the MIC. Results are representative of two to three separate experiments.

FIG. 2

Permeabilization of the cytoplasmic membrane of S. aureus as a function of peptide concentration, indicated by maximum fluorescence reached within 5 min. Shown are results with CP11CN (▴), indolicidin (●), CP29 (▾), CP26 (■), and Bac2A-NH₂ (+). Results are representative of two to three separate experiments.

FIG. 3

Permeabilization of the cytoplasmic membrane of S. aureus (dashed lines) as indicated by the kinetics of fluorescence intensity changes in the presence of 8 μg of CP26 (▴) per ml or 2 μg of CP29 (●) per ml (A) and in the presence of 32 μg of indolicidin (▴) or CP11CN (●) per ml (B). Shown are the levels of survival (in CFU per milliliter) of bacteria under the dye assay conditions (solid lines). Results are representative of two to three separate experiments.

FIG. 4

Electron micrographs of untreated (top left), CP29-treated (top right), CP11CN-treated (bottom left), and Bac2A-NH₂-treated (bottom right) S. aureus (A) and S. epidermidis (B). All peptides were at concentrations 10 times the MIC. The bar is equal to 250 nm.

FIG. 4

Electron micrographs of untreated (top left), CP29-treated (top right), CP11CN-treated (bottom left), and Bac2A-NH₂-treated (bottom right) S. aureus (A) and S. epidermidis (B). All peptides were at concentrations 10 times the MIC. The bar is equal to 250 nm.