Single-cell, time-resolved study of the effects of the antimicrobial peptide alamethicin on Bacillus subtilis

Abstract

Alamethicin is a well-studied antimicrobial peptide (AMP) that kills Gram-positive bacteria. It forms narrow, barrel-stave pores in planar lipid bilayers. We present a detailed, time-resolved microscopy study of the sequence of events during the attack of alamethicin on individual, live Bacillus subtilis cells expressing GFP in the cytoplasm. At the minimum inhibitory concentration (MIC), the first observed symptom is the halting of growth, as judged by the plateau in measured cell length vs time. The data strongly suggest that this growth-halting event occurs prior to membrane permeabilization. Gradual degradation of the proton-motive force, inferred from a decrease in pH-dependent GFP fluorescence intensity, evidently begins minutes later and continues over about 5 min. There follows an abrupt permeabilization of the cytoplasmic membrane to the DNA stain Sytox Orange and concomitant loss of small osmolytes, causing observable cell shrinkage, presumably due to decreased turgor pressure. This permeabilization of the cytoplasmic membrane occurs uniformly across the entire membrane, not locally, on a timescale of 5s or less. GFP gradually leaks out of the cell envelope, evidently impeded by the shrunken peptidoglycan layer. Disruption of the cell envelope by alamethicin occurs in stages, with larger and larger species permeating the envelope as time evolves over tens of minutes. Some of the observed symptoms are consistent with the formation of barrel-stave pores, but the data do not rule out "chaotic pore" or "carpet" mechanisms. We contrast the effects of alamethicin and the human cathelicidin LL-37 on B. subtilis.

Keywords: Alamethicin; Antimicrobial peptide; Bacillus subtilis; Barrel-stave pore; Fluorescence microscopy; Membrane permeabilization; Real-time imaging; Single-cell imaging.

Figure 1

Left: Three channel montage of images vs time for a single B. subtilis cell exposed to 16 µg/mL alamethicin (1X MIC) and 2 nM Sytox Orange beginning at t = 0. Fluorescence images of cytoplasmic GFP and Sytox Orange and phase contrast images were obtained at times shown. Scale bar is 2 µm. Right: For the same cell, plots of total GFP intensity (green circles), total Sytox Orange intensity (orange squares), and cell length (black triangles) vs time. Arrows point to the appropriate axis for each plot. Only one single cell was amenable to quantitative analysis.

Figure 2

(A) Normalized total GFP fluorescence intensity vs time for 19 cells exposed to 16 µg/mL alamethicin (thin gray lines). For phasing, this plot defines time t’ = 0 as the end of the abrupt cell shrinkage event. The average of all 19 traces is shown as the heavy black line. The time of alamethicin injection varies from t’ = –17.1 to –7.5 min. The average decrease in GFP intensity is 20%. (B) Normalized total GFP fluorescence intensity for 11 cells treated with 5 µM nigericin at t = –6 min. At t = 0 the flow was changed to 5 µM nigericin plus 16 µg/mL alamethicin. The average of all 11 traces is shown as the heavy black line. The nigericin-induced decrease in GFP intensity from plateau to plateau is ~30%.

Figure 3

Left: Three channel montage of images vs time for a representative double B. subtilis cell exposed to 16 µg/mL alamethicin (1X MIC) and 2 nM Sytox Orange beginning at t = 0. Fluorescence images of cytoplasmic GFP and Sytox Orange and phase contrast images were obtained at times shown. Scale bar is 2 µm. Right: For the same cell, plots of total GFP intensity (green circles), total Sytox Orange intensity (orange squares), and cell length (black triangles) vs time. Arrows point to the appropriate axis for each plot. The circled region highlights the time between the first and second abrupt cell shrinkage events. Eighteen of the 19 cells analyzed were double cells with behavior comparable to that shown here.

Figure 4

Example of cell length and GFP intensity vs time plot for one double cell. Cell length flattens rather abruptly well before the GFP intensity begins to decrease. The construction shown is used to measure the lag time between the two events; Δt = 2.7 min in this case. See Fig. S4 for additional examples, one of which exhibits transitions that are less sharp in time.

Figure 5

Distribution of lag times between abrupt cell shrinkage and the onset of complete loss of GFP from the cell envelope. Black bars are for 18 cells exposed to 16 µg/mL alamethicin. Cross-hatched bars are for 17 cells exposed to 16 µg/mL alamethicin plus lysozyme at 0.2 µg/mL, with lysozyme added t = 5 min, after cell shrinkage had occurred. The mean value of (t_GFP – t_shrink) is 34 ± 11 min with alamethicin alone and 20 ± 5 min with alamethicin plus lysozyme (± 1 standard deviation).

Figure 6

Comparison of dynamics of nucleoid staining by Sytox Orange exposed to 2 µM LL-37 (top, 2X MIC) vs 16 µg/mL alamethicin (bottom, 1X MIC). A single phase contrast image was taken before the start of the Sytox Orange data acquisition, as shown at left. Data were taken at 0.5 sec/frame; every 10^th frame is shown.