Interactions of bacterial cationic peptide antibiotics with outer and cytoplasmic membranes of Pseudomonas aeruginosa

Abstract

Polymyxins B and E1 and gramicidin S are bacterium-derived cationic antimicrobial peptides. The polymyxins were more potent than gramicidin S against Pseudomonas aeruginosa, with MICs of 0.125 to 0. 25 and 8 microg/ml, respectively. These peptides differed in their affinities for binding to lipopolysaccharide, but all were able to permeabilize the outer membrane of wild-type P. aeruginosa PAO1 strain H103, suggesting differences in their mechanisms of self-promoted uptake. Gramicidin S caused rapid depolarization of the bacterial cytoplasmic membrane at concentrations at which no killing was observed within 30 min, whereas, conversely, the concentrations of the polymyxins that resulted in rapid killing resulted in minimal depolarization. These data indicate that the depolarization of the cytoplasmic membrane by these peptides did not correlate with bacterial cell lethality.

FIG. 1

Influence of peptide addition to the aqueous subphase bathing the LPS monolayers on the surface pressure, as measured in a Langmuir balance. Peptide (0.8 μg/ml [0.32 μg/ml in the case of gramicidin S]) was added to the aqueous subphase bathing the S. enterica serovar Minnesota Re LPS monolayers. The Mg²⁺ concentration of the subphase was varied from 0 (filled bars) to 2 mM (open bars) to 5 mM (hatched bars). The results shown are the averages of two independent experiments.

FIG. 2

Relationship between cytoplasmic membrane permeabilization, as assessed by the diSC₃5 assay, and cell killing, as measured by determination of the numbers of CFU at the same time as the permeabilization assay. Dotted curves represent data obtained from the diSC35 assay, and solid curves represent data obtained from killing assays. ●, polymyxin E1 at 2 μg/ml; ○, polymyxin E1 at 10 μg/ml; ▾, gramicidin S at 8 μg/ml; ▵, polymyxin B at 2 μg/ml; ▴, colymycin M at 64 μg/ml. Abbreviations: PXB, polymyxin B; PXE1, polymyxin E1; GS, gramicidin S; CM, colymycin M. The number following the abbreviation is the concentration applied (in micrograms per milliliter).

FIG. 3

Influence of gramicidin S (GS), polymyxin B (PXB), polymyxin E1 (PXE1), or colymycin M (CM) addition to the aqueous subphase on the surface pressure of phospholipid monolayers, as measured in a Langmuir balance. (A) Plot of the surface pressure increase as a function of peptide concentration. Titration of the surface pressure increase was accomplished by adding successive amounts of peptide to the subphase while continuously monitoring the surface pressure of the film. (B) Influence on surface pressure of the addition of 1 μg of a peptide per ml to the aqueous phase of a monolayer made from POPC, POPE, egg-PG, or CL. The results shown are the averages of two independent experiments.