Depolarization, bacterial membrane composition, and the antimicrobial action of ceragenins

Abstract

Ceragenins are cholic acid-derived antimicrobial agents that mimic the activity of endogenous antimicrobial peptides. Ceragenins target bacterial membranes, yet the consequences of these interactions have not been fully elucidated. The role of the outer membrane in allowing access of the ceragenins to the cytoplasmic membrane of gram-negative bacteria was studied using the ML-35p mutant strain of Escherichia coli that has been engineered to allow independent monitoring of small-molecule flux across the inner and outer membranes. The ceragenins CSA-8, CSA-13, and CSA-54 permeabilize the outer membrane of this bacterium, suggesting that the outer membrane does not play a major role in preventing the access of these agents to the cytoplasmic membrane. However, only the most potent of these ceragenins, CSA-13, was able to permeabilize the inner membrane. Interestingly, neither CSA-8 nor CSA-54 caused inner membrane permeabilization over a 30-min period, even at concentrations well above those required for bacterial toxicity. To further assess the role of membrane interactions, we measured membrane depolarization in gram-positive bacteria with different membrane lipid compositions, as well as in gram-negative bacteria. We found greatly increased membrane depolarization at the minimal bactericidal concentration of the ceragenins for bacterial species containing a high concentration of phosphatidylethanolamine or uncharged lipids in their cytoplasmic membranes. Although membrane lipid composition affected bactericidal efficiency, membrane depolarization was sufficient to cause lethality, providing that agents could access the cytoplasmic membrane. Consequently, we propose that in targeting bacterial cytoplasmic membranes, focus be placed on membrane depolarization as an indicator of potency.

FIG. 1.

Structures of ceragenins CSA-8, CSA-13, and CSA-54.

FIG. 2.

Outer and inner membrane permeabilization of E. coli ML-35p caused by one of the ceragenins or by melittin as a function of time at different concentrations (0 to 100 μg/ml) and at 37°C. Hydrolysis of nitrocefin by β-lactamase was used to monitor outer membrane permeabilization by absorbance at 486 nm (left-hand panel). Hydrolysis of ONPG by β-galactosidase was used to monitor inner membrane permeabilization by absorbance at 420 nm (right-hand panel).

FIG. 3.

Effects of CSA-13 on the fluorescence intensity of DiS-C₂(5) in the presence of the indicated organisms. (A) S. aureus ATCC 25923. CSA-13 was added at 90 s at the following concentrations: line 1, 0.125 μg/ml; line 2, 0.2.5 μg/ml; line 3, 5.0 μg/ml; and line 4, 10.0 μg/ml. (B) B. cereus ATCC 11778. CSA-13 was added at 90 s at the following concentrations: line 1, 0.5 μg/ml; line 2, 1.0 μg/ml; line 3, 2.0 μg/ml; and line 4, 4.0 μg/ml. cps, counts per second.

FIG. 4.

Percentage of membrane depolarization with the indicated organisms at 330 s determined as an increase in fluorescence intensity of DiS-C₂(5) and compared to the maximum fluorescence increase at 4× MBC for CSA-13. White bars, CSA-13; black bars, CSA-54; gray bars, CSA-8.

FIG. 5.

Effects of EDTA and ceragenins CSA-8 and CSA-13 on the fluorescence intensity of DiS-C₂(5) in the presence of the indicated organisms. The ceragenins and EDTA were added at 3,600 s. (A) EDTA, at the indicated concentrations, with K. pneumoniae ATCC 13883. (B) CSA-8, at the indicated concentrations, with K. pneumoniae ATCC 13883. (C) CSA-13, at the indicated concentrations, with K. pneumoniae ATCC 13883. (D) CSA-13, at the indicated concentrations, with E. coli ATCC 25922.