Stages of polymyxin B interaction with the Escherichia coli cell envelope

Abstract

The effects of polymyxin B (PMB) on the Escherichia coli outer (OM) and cytoplasmic membrane (CM) permeabilities were studied by monitoring the fluxes of tetraphenylphosphonium, phenyldicarbaundecaborane, and K(+) and H(+) ions. At concentrations between 2 and 20 microgram/ml, PMB increased the OM permeability to lipophilic compounds and induced a leakage of K(+) from the cytosol and an accumulation of lipophilic anions in the cellular membranes but did not cause the depolarization of the CM. At higher concentrations, PMB depolarized the CM, forming ion-permeable pores in the cell envelope. The permeability characteristics of PMB-induced pores mimic those of bacteriophage- and/or bacteriocin-induced channels. However, the bactericidal effect of PMB took place at concentrations below 20 microgram/ml, indicating that this effect is not caused by pore formation. Under conditions of increased ionic strength, PMB made the OM permeable to lipophilic compounds and decreased the K(+) gradient but was not able to depolarize the cells. The OM-permeabilizing effect of PMB can be diminished by increasing the concentration of Mg(2+). The major new findings of this work are as follows: (i) the OM-permeabilizing action of PMB was dissected from its depolarizing effect on the CM, (ii) the PMB-induced ion-permeable pores in bacterial envelope were registered, and (iii) the pore formation and depolarization of the CM are not obligatory for the bactericidal action of PMB and dissipation of the K(+) gradient on the CM.

FIG. 1

Effects of PMB, PMBN, and EDTA on the accumulation of TPP⁺ (A and B) and PCB⁻ (C and D) by E. coli AN180 (A and C) and KO1489 (B and D) cells. The experiments were performed at 37°C in 100 mM Tris-HCl (pH 8.0). The cell concentration was 3 × 10⁹ cells/ml. Arrows, if not stated otherwise, indicate the addition of PMB (curve 1), PMBN (curve 2), or EDTA (curve 3). The number next to the arrow indicates the final concentration (in micrograms per milliliter) of PMB or PMBN after the last addition. EDTA was added to 0.03, 0.12, 1, and 5 mM (A and C), and GD was added to 5 μg/ml. The results shown are representative of three independent experiments.

FIG. 2

Effects of PMB and PMBN on accumulation of TPP⁺ (A and B) and PCB⁻ (C and D) by E. coli AN180 (A and C) and KO1489 (B and D) cells. The initial conditions of the experiments were as described in the legend to Fig. 1, but the experiments were performed in 100 mM sodium phosphate (pH 8.0). Arrows, if not stated otherwise, indicate the addition of PMB (curve 1) and PMBN (curve 2). A number next to the arrow indicates the final concentration (in micrograms per milliliter) of PMB or PMBN after the last addition. GD was added to a concentration of 5 μg/ml. The results shown are representative of three independent experiments.

FIG. 3

Effect of PMB on the viabilities of E. coli AN180 (○) and KO1489 (●) cells. The viabilities of the cells were measured as described in Materials and Methods, using 100 mM sodium phosphate (pH 8.0) as the incubation medium. Each datum point represents the mean of values from three independent experiments. The standard errors of the means were all less than 10%.

FIG. 4

Effect of Mg²⁺ on PMB-induced TPP⁺ uptake by E. coli AN180 (A) or KO1489 (B) cells. The experiments were performed at 37°C in 100 mM Tris-HCl (pH 8.0), and MgCl₂ was added to the concentrations (in millimolar) indicated in the figure. The cells were added to a final concentration of 3 × 10⁹ cells/ml, and GD was added to a final concentration of 5 μg/ml. (A) Arrows, if not stated otherwise, indicate the addition of PMB, and a number next to the arrow indicates the final concentration (in micrograms per milliliter) of PMB after the last addition. (B) PMB was added to a concentration 80 μg/ml. The results shown are representative of three independent experiments.

FIG. 5

Effect of Mg²⁺ on the viability of PMB-treated cells. The viability of the cells was measured as indicated in Materials and Methods, using 100 mM Tris-HCl (pH 8.0) as the incubation medium. ●, experiments carried out in the absence of Mg²⁺; ○, experiments carried out in the presence of 40 mM Mg²⁺; ▾, experiments carried out in the presence of 40 mM Mg²⁺ but Mg²⁺ was not included in the 0.9% NaCl solutions used for the dilutions before plating. (A) AN180 cells; (B) Tris-EDTA-treated AN180 cells; (C) KO1489 cells. Each data point represents the mean of values from three independent experiments. The standard errors of the means were all less than 10%.

FIG. 6

Effects of PMB and PMBN on the efflux of K⁺ from E. coli cells. The experiments were performed at 37°C (A and C) or 25°C (B) in buffers consisting of 100 mM sodium phosphate at pH 8.0 (A, curves 1 to 3) or pH 7.0 (A, curve 4) or in 100 mM Tris-HCl at pH 8.0 (B and C). In panel B, curves 3 and 4, and panel C, curve 3, the incubation medium contained 8 mM Mg²⁺. The cell concentration was 3 × 10⁹ cells/ml, and GD (A and C) was added to 5 μg/ml. Arrows, if not stated otherwise, indicate the addition of PMBN (A, curves 3 and 4) or PMB. The number next to the arrow indicates the final concentration (in micrograms per milliliter) of PMB or PMBN after the last addition. Curve 2 in each panel and curve 4 in panel B are data from experiments with KO1489 cells; the rest of the experiments were carried out with AN180 cells. The results shown are representative of three independent experiments.

FIG. 7

KO1489 (A) and AN180 (B) cell sensitivity to PMBN. The viabilities of the cells were measured as indicated in Materials and Methods. The incubation media were 100 mM Tris-HCl (pH 8.0) (in panel A, ○; in panel B, ○ and ●, 100 mM Tris-HCl (pH 7.0) (in panel A, ●), and 100 mM sodium phosphate (pH 8.0) (in panel A, ▾). (B) ○, Tris-EDTA-treated cells; ●, nontreated cells. Each datum point represents the mean of values from three independent experiments. The standard errors of the means were all less than 10%.

FIG. 8

Effect of PMB on the pH of the bacterial suspension. The experiment was performed at 32°C. The incubation medium contained 0.5 mM MOPS in 100 mM NaCl (pH adjusted by Tris to 6.75) and 3 × 10⁹ AN180 cells/ml. Arrows, if not stated otherwise, indicate the addition of PMB, and a number next to the arrow indicates the final concentration (in micrograms per milliliter) of PMB after the last addition. GD was added to a final concentration of 5 μg/ml. The results shown are representative of three independent experiments.

FIG. 9

Effects of PMB, phage T4, and GD on the E. coli respiration-driven proton pump. The anaerobic incubation medium (pH 6.75) contained 0.5 mM MOPS, 100 mM NaCl (see Fig. 8), 3 × 10⁹ AN180 cells/ml, and 100 μg of PMB per ml (A), 12 × 10⁹/ml infectious particles of phage T4 (B), or 10 μg of GD per ml (C). Oxygen pulse (added as air-saturated H₂O) contained 8.25 nmol of O₂. The temperature of the medium was 32°C (A and B, curves 1 and 2, and C, curve 1) or 10°C (A and B, curve 3, and C, curve 2). The experiment was repeated three times with similar results, and the data from one representative experiment are shown.