Functional interrelationships between cell membrane and cell wall in antimicrobial peptide-mediated killing of Staphylococcus aureus

Abstract

Perturbation of the Staphylococcus aureus cytoplasmic membrane (CM) is felt to play a key role in the microbicidal mechanism of many antimicrobial peptides (APs). However, it is not established whether membrane permeabilization (MP) alone is sufficient to kill susceptible staphylococci or if the cell wall (CW) and/or intracellular targets contribute to AP-induced lethality. We hypothesized that the relationships between MP and killing may differ for distinct APs. In this study, we investigated the association between AP-induced MP and lethality in S. aureus whole cells versus CW-free protoplasts, and in comparison to the MP of liposomes modeled after whole CMs in terms of phospholipid composition, fluidity and charge. Four APs with different structure-activity relationships were examined: thrombin-induced platelet microbicidal protein 1 (tPMP-1), human neutrophil protein 1 (hNP-1), gramicidin D, and polymyxin B. MP was quantified fluorometrically by calcein release. All APs tested, except polymyxin B, caused concentration-dependent MP and killing of whole cells, but not of protoplasts. The reduced AP susceptibility of protoplasts was associated with increased cardiolipin and lysyl-phosphatidylglycerol content and reduced fluidity of their CMs. However, liposomal MP induced by tPMP-1, hNP-1, and gramicidin D paralleled that of whole cells. Collectively, these results indicate that (i) structurally distinct APs likely exert their staphylocidal effects by differing mechanisms, (ii) MP is not the sole event leading to AP-induced staphylocidal activity, (iii) a complex interrelationship exists between the CM and CW in AP-induced killing, and (iv) liposomes modeled upon whole cell or protoplast CMs can recapitulate the respective susceptibilities to killing by distinct APs.

FIG. 1.

(A) Staphylocidal activity of antimicrobial peptides on S. aureus whole cells. Killing of S. aureus whole cells exposed to different concentrations (as discussed in Materials and Methods) of antimicrobial peptides for 1 h with respect to the untreated control cells is shown. hNP-1 (□) tPMP-1 (), gramicidin D (), and polymyxin B (▪) were used in this study. Each experiment was done in triplicate, and the values represent means ± standard deviations. (B) Kinetics of staphylocidal activity. Killing of S. aureus over time following exposure to tPMP-1 at 0.5 μg/ml (•), 1.0 μg/ml (▪), or 2.0 μg/ml (▴) and gramicidin D at 1× MIC (•), 2.5× MIC (▪), 5× MIC (▴), or 10× MIC (). (C) S. aureus whole-cell membrane permeabilization by antimicrobial peptides. Membrane permeabilization of S. aureus whole cells by antimicrobial peptides for 1-h exposure was detected via the leakage of calcein. hNP-1 (□), tPMP-1 (), gramicidin D (), and polymyxin B (▪) were used in this study. Each experiment was done in triplicate, and the values represent means ± standard deviations. (D) Kinetics of membrane permeabilization. Membrane permeabilization of S. aureus over time following exposure to tPMP-1 at 0.5 μg/ml (•), 1.0 μg/ml (▪), or 2.0 μg/ml (▴) and gramicidin D at 1× MIC (•), 2.5× MIC (▪), 5× MIC (▴), or 10× MIC (). (E) Correlation analysis of the relationship between killing (−Δlog₁₀ CFU/ml) and membrane permeabilization (% calcein leakage). Linear regressions were determined, and correlation coefficients (r²) were calculated from mean results of three independent experiments. r² for tPMP-1 (⧫), hNP-1 (□), and gramicidin D (▵) are 0.9925, 0.9961, and 0.8074, respectively (P < 0.05 for each).

FIG. 1.

(A) Staphylocidal activity of antimicrobial peptides on S. aureus whole cells. Killing of S. aureus whole cells exposed to different concentrations (as discussed in Materials and Methods) of antimicrobial peptides for 1 h with respect to the untreated control cells is shown. hNP-1 (□) tPMP-1 (), gramicidin D (), and polymyxin B (▪) were used in this study. Each experiment was done in triplicate, and the values represent means ± standard deviations. (B) Kinetics of staphylocidal activity. Killing of S. aureus over time following exposure to tPMP-1 at 0.5 μg/ml (•), 1.0 μg/ml (▪), or 2.0 μg/ml (▴) and gramicidin D at 1× MIC (•), 2.5× MIC (▪), 5× MIC (▴), or 10× MIC (). (C) S. aureus whole-cell membrane permeabilization by antimicrobial peptides. Membrane permeabilization of S. aureus whole cells by antimicrobial peptides for 1-h exposure was detected via the leakage of calcein. hNP-1 (□), tPMP-1 (), gramicidin D (), and polymyxin B (▪) were used in this study. Each experiment was done in triplicate, and the values represent means ± standard deviations. (D) Kinetics of membrane permeabilization. Membrane permeabilization of S. aureus over time following exposure to tPMP-1 at 0.5 μg/ml (•), 1.0 μg/ml (▪), or 2.0 μg/ml (▴) and gramicidin D at 1× MIC (•), 2.5× MIC (▪), 5× MIC (▴), or 10× MIC (). (E) Correlation analysis of the relationship between killing (−Δlog₁₀ CFU/ml) and membrane permeabilization (% calcein leakage). Linear regressions were determined, and correlation coefficients (r²) were calculated from mean results of three independent experiments. r² for tPMP-1 (⧫), hNP-1 (□), and gramicidin D (▵) are 0.9925, 0.9961, and 0.8074, respectively (P < 0.05 for each).

FIG. 2.

(A) Staphylocidal activity of antimicrobial peptides on S. aureus protoplasts. Killing of S. aureus protoplasts exposed to different concentrations of antimicrobial peptides for 1 h with respect to the untreated control is shown. (B) Membrane permeabilization by antimicrobial peptides on S. aureus protoplasts. Membrane permeabilization of S. aureus protoplasts by antimicrobial peptides for 1-h exposure was detected via the leakage of calcein. hNP-1 (□), tPMP-1 (), gramicidin D (), and polymyxin B (▪) were used in this study. Each experiment was done in triplicate, and the values represent means ± standard deviations.

FIG. 3.

Membrane characteristics of whole cells versus protoplasts versus liposomes. (A) Phospholipid composition of whole cells (□) and protoplasts (▪). Phospholipid contents were determined by 2D-TLC and expressed as percentages of total phospholipids as described in Materials and Methods. (B) Membrane fluidity of whole cells (□) and protoplasts (▪) and of liposomes of different composition (C) was determined by fluorescence polarization (p) measurements using fluorescent probe DPH as described in Materials and Methods. Each estimation was done at least in triplicate, and the values represent means ± SDs.

FIG. 4.

Liposomal permeabilization by antimicrobial peptides. Liposomes with different phospholipid formulations (in molar ratio) of DPPG:CL (20:1) (A), SA:DPPG:CL (1:9:2) (B), and SA:DPPG:CL (1:4:1) (C) were incubated at 37°C with different APs, and permeabilization was measured via calcein leakage. hNP-1 (□), tPMP-1 (), gramicidin D (), and polymyxin B (▪) were used in this study. Each experiment was done in triplicate, and the values represent means ± SDs.