Inhibitory action of a truncated derivative of the amphibian skin peptide dermaseptin s3 on Saccharomyces cerevisiae

Abstract

The inhibitory activity of a truncated derivative of the natural amphibian skin peptide dermaseptin s3-(1-16)-NH2 [DS s3 (1-16)] against Saccharomyces cerevisiae was studied. Significant growth inhibition was observed after exposure to 3.45 microgram of the peptide per ml at pH 6.0 and 7.0, with complete growth inhibition occurring at 8.63 microgram of peptide per ml for all pH values tested. Using confocal scanning laser microscopy, we have shown that DS s3 (1-16) disrupted the yeast cell membrane resulting in the gross permeabilization of the cell to the nuclear stain ethidium bromide. However, the principal inhibitory action of the peptide was not due to disruption of intracellular pH homeostasis. Instead, growth inhibition by the peptide correlated with the efflux of important cellular constituents such as ADP, ATP, RNA, and DNA into the surrounding medium. The combination of DS s3 (1-16) with mild heating temperatures as low as 35 degreesC significantly enhanced the inhibitory effect of the peptide (8.63 microgram/ml), and at 45 degreesC greater than 99% of the population was killed in 10 min. In summary, a derivative of a natural antimicrobial peptide has potential, either alone or in combination with mild heating, to prevent the growth of or kill spoilage yeast.

FIG. 1

Effect of 0 (⧫), 0.86 (◊), 1.73 (▴), 3.45 (□), and 8.63 (○) μg of DS s3 (1-16) per ml on the growth of S. cerevisiae X2180-1A in YEPD (inoculum size, 5.0 × 10³ cells ml⁻¹) at pH 7.0 (a), pH 6.0 (b), and pH 4.0 (c). Growth was measured turbidometrically at 600 nm at 30°C with shaking over a period of 7 days. A representative result of at least three replicate experiments is shown.

FIG. 2

Effect of 8.63 μg of DS s3 (1-16) per ml on viability over a 7-h incubation of a mid-exponential-phase culture (5.0 × 10⁷ cells ml⁻¹) of S. cerevisiae X2180-1A in YEPD at 30°C at pH 3.0 (▵), pH 4.0 (■), pH 5.0 (○), pH 6.0 (⧫), and pH 7.0 (□). A representative result of at least three replicate experiments is shown.

FIG. 3

Effect of inoculum sizes of 5.0 × 10³ cells ml⁻¹ (□, ■), 5.0 × 10² cells ml⁻¹ (◊, ⧫), and 5.0 × 10 cells ml⁻¹ (○, •) on the growth of S. cerevisiae X2180-1A in YEPD at pH 6.0 either without (open symbols) or in the presence of (solid symbols) 1.73 μg of DS s3 (1-16) per ml. Growth was measured turbidometrically at 600 nm at 30°C with shaking over a period of 7 days. A representative result of at least three replicate experiments is shown.

FIG. 4

Results of a time course experiment illustrating the effect of exposure to 12.97 μg of DS s3 (1-16) per ml on the permeability of the membrane of an individual cell of S. cerevisiae X2180-1A in YEPD (pH 6.0) by CSLM. Peptide-induced membrane disruption was visualized by influx of the fluorescent nuclear stain ethidium bromide (10 μg/ml) over a time course of 13 s. The captured images are representative of a typical result.

FIG. 5

Effect of a 10-min exposure to increasing concentrations of DS s3 (1-16) (0 to 103.62 μg/ml) on the membrane permeability (■) and viability (□) of a mid-exponential-phase culture of S. cerevisiae X2180-1A in YEPD at pH 6.0. Peptide-induced permeability to ethidium bromide (10 μg/ml) was measured from captured fluorescent images (obtained by CSLM) and was expressed as the percentage of fluorescent cells in the population. Each datum point represents the mean and standard deviation acquired from the counting of at least 300 randomly captured cells.

FIG. 6

Effect of exposure to DS s3 (1-16) on the pH_i of S. cerevisiae X2180-1A. The effect of 0 (■, □), 8.63 (⧫, ◊) and 17.27 (▴, ▵) μg of DS s3 (1-16) per ml on the pH_i (solid symbols) of cells growing in YNBG at pH 5.0 (open symbols) was determined. The arrow and dotted line indicate when the peptide was added to the cultures. Growth was measured turbidometrically at 600 nm, and the pH_i was measured by determining the intracellular pH-dependent fluorescence of CF-SE. Representative results of at least two independent experiments are shown.

FIG. 7

Comparison of the levels of extracellular ATP or ADP effluxed from growing cultures of S. cerevisiae X2180-1A at pH 4.5 (a) and pH 6.0 (b) before (preaddition) and after (35 and 170 min postaddition) exposure to 17.27 μg of DS s3 (1-16) per ml. Extracellular ATP (▤) or ADP (□) levels during growth in the presence of 17.27 μg of DS s3 (1-16) per ml were compared to the levels of extracellular ATP (■) or ADP (▩) without the presence of the peptide. Representative results of at least two independent experiments are shown.

FIG. 8

Effect of exposure to 17.27 μg of DS s3 (1-16) per ml on the efflux of UV-absorbing compounds from S. cerevisiae X2180-1A. The arrow and dotted line indicate when the peptide was added to the cultures. Efflux of UV-absorbing compounds was measured from cells resuspended in 25 mM citric-phosphate buffer at pH 4.5 (▴, ▵) and pH 6.0 (■, □). Efflux was determined in the presence of the peptide (open symbols) or without peptide (solid symbols) by measuring the optical density at 260 nm (adjusted for the presence of the peptide) and was expressed as the A₂₆₀ of the cell-free solution. A representative result is shown.

FIG. 9

Effect of exposure to 34.54 μg of DS s3 (1-16) per ml on the membrane permeability of mid-exponential-phase cells of S. cerevisiae X2180-1A in YEPD at pH 6.0 (■). After 25 min of exposure, the treated cells were removed by centrifugation and the supernatant was retained and subsequently used to challenge a fresh, identical population of cells for a further 25 min (□). Peptide-induced permeability of the cell to ethidium bromide (10 μg/ml) was measured from captured fluorescent images (obtained by CSLM) and was expressed as the percentage of fluorescent cells in the population. Each datum point represents the mean and standard deviation acquired from the counting of at least 300 randomly captured cells.

FIG. 10

Effect of increasing temperature (25 to 45°C) on the membrane permeability (solid symbols) (a) and viability (open symbols) (b) of mid-exponential-phase cells of S. cerevisiae X2180-1A (YEPD, pH 6.0) exposed to 0 (■, □), 3.45 (▴, ▵) and 8.63 (○, •) μg of DS s3 (1-16) per ml for 10 min. Membrane permeability was measured by fluorescence microscopy as described in the text, and each datum point represents the mean and standard deviation acquired from the counting of at least 300 randomly captured cells.