Mode of action and membrane specificity of the antimicrobial peptide snakin-2

Microbial strains, cell lines, and media

For the microdilution assay, time-kill curves, and growth-inhibition curves, a gram-positive bacterial strain (Bacillus subtilis), a gram-negative strain (Escherichia coli), and a yeast (Saccharomyces cerevisiae) were chosen to characterize the antimicrobial activity of SN2. These organisms are representatives of three major groups of microbes. We used these strains to get an overview of the antimicrobial activity among different microorganisms. Many food-spoiling organisms belong to the group of filamentous fungi, but we excluded this group in this study, because it was characterized in our last study with the representative Fusarium solani (Herbel, Schäfer & Wink, 2015). The bacteria were grown at 37 °C in salt-free Luria-Bertani (LB) broth (1% tryptone, 0.5% yeast extract), and the yeast at 28 °C in Sabouraud-Glucose (SAB) broth (2% mycological peptone, 4% D(+)-glucose). 1.5% agar was added to prepare solid media. The pore-forming activity of SN2 in biomembranes was observed using protoplasts derived from a suspension culture of Nicotiana tabacum grown in Murashige and Skoog (MS) liquid medium (Murashige & Skoog, 1962) but without kinetin and indolylacetic acid.

Recombinant expression of SN2 in E. coli

The SN2 peptide was expressed in E. coli BL21[DE3] as previously described (Herbel, Schäfer & Wink, 2015). Purification was achieved by affinity chromatography using the Äkta start system (GE Healthcare, Solingen, Germany) combined with Bio-Scale Mini Profinity IMAC Cartridges (Bio-Rad, Munich, Germany). The SN2 peptide was expressed as a protein fused to thioredoxin to mask the antimicrobial activity during expression in the bacterial host. After purification of the fusion protein, thioredoxin was removed by TEV protease digestion (Herbel, Schäfer & Wink, 2015).

Microdilution

The microdilution assay to determine MIC (minimal inhibitory concentration) of recombinant SN2 was used as previously described (Herbel, Schäfer & Wink, 2015). Liquid growth media and microbial suspensions of 1 × 10⁶ cfu/ml (bacteria) or 5 × 10⁵ cfu/ml (yeast) were added to serially diluted SN2 (35-0.01 µM) and incubated for 24 h at 37 °C (bacteria) or 28 °C (yeast). MIC was determined as the lowest concentration without visible growth.

Time-kill assay

The bactericidal activity of snakin-2 was analyzed using time-kill curves. For these experiments, recombinant SN2 was added in final concentrations of 0.25×, 0.5×, 1×, and 2× of its MIC to bacterial (5 × 10⁵ cfu/ml) or yeast (2.5 × 10⁵ cfu/ml) cultures, and aliquots were taken after 0, 0.5, 1, 3, 6, and 24 h. These were diluted with the respective liquid medium and plated on agar plates. After incubation for 24 h at 37 °C (bacteria) or 28 °C (yeast), the colonies were counted.

Growth inhibition assay

The inhibitory effect of SN2 on the growth of microorganisms was determined by growth inhibition curves. SN2 was serially diluted (1× MIC – 1/32× MIC) and bacterial (5 × 10⁵ cfu/ml) or yeast (2.5 × 10⁵ cfu/ml) cultures were added. After 0, 3, 6, 7.5, 9, and 24 h, aliquots were taken, and the optical density was measured at 600 nm using a spectrophotometer (WPA Biowave II, Biochrom, Cambridge, UK).

Salt sensitivity assay

Since AMPs can be salt dependent, this assay was used to determine the inhibition of SN2 activity in the presence of monovalent cations under more physiological conditions. Recombinant SN2 was used in its 1 × MIC, and NaCl or KCl was added to obtain final concentrations of 0, 25, 50, 100, 150, and 300 mM respectively. Bacterial (5 × 10⁵ cfu/ml) or yeast (2.5 × 10⁵ cfu/ml) cultures were added, and the growth was measured as optical density at 600 nm after 9 h. The growth of a control culture (without SN2) was set to 100% growth and consequential as 0% activity. No growth was set to 100% activity of SN2.

Hemolysis and hemagglutination assay

To estimate a potential risk for humans, if SN2 would be applied as a pharmaceutical drug, the activity of SN2 was tested on mammalian erythrocytes as a proxy for human cells. To study a potential hemolytic and hemagglutinating effect of SN2, 100 µl red blood cells (defibrinated sheep blood, Thermo Scientific, Braunschweig, Germany) were washed three times with 0.4 M mannitol and mixed in a 96-well plate with 100 µl serially diluted SN2 (35-0.01 µM). 0.4 M mannitol and 1% SDS were used respectively as negative (0% hemolysis) and positive controls (100% hemolysis). After incubation at 26 °C for 1 h, the lowest SN2 concentration that caused distinct hemagglutination was visually identified. After centrifugation, the absorbance of the supernatant was determined by measuring the absorption spectrophotometrically at 570 nm. The percentage of hemolysis was calculated as $100^{\ast }\left({\mathrm{A}}_{SN2}-{\mathrm{A}}_{\text{mannitol}}\right)∕\left({\mathrm{A}}_{SDS}-{\mathrm{A}}_{\text{mannitol}}\right)$ (Ramirez-Carreto et al., 2015).

Protoplast production and microscopy

Protoplasts were used to analyze the activity against plant membranes to further understand the behavior of SN2 in its host cells and to study the membrane specificity of SN2. Thereby, the potential of SN2 as a pharmaceutical drug can be evaluated. Protoplasts were produced from a suspension cell culture of Nicotiana tabacum 4 days after subculturing. 1% cellulase and 0.25% macerozyme were added to 15 ml of the suspension culture and incubated in a petri dish for 2 h at 26 °C, shaking circumspectly. The protoplasts were washed three times with 0.4 M mannitol and used for microscopy (Keyence BZ-9000, KEYENCE Deutschland GmbH, Neu-Isenburg, Germany). SN2 (17 µM final concentration) was added to the protoplasts or undigested tobacco suspension cells, respectively, and photographs were taken with the microscope software (BZII Viewer, KEYENCE Deutschland GmbH, Neu-Isenburg, Germany).

All experiments were repeated three times. In the graphs, the standard deviations from three repetitions are shown as error bars.

Bactericidal and fungicidal kinetics of SN2

Bactericidal activity against a gram-negative and gram-positive bacterial strain and fungicidal effects against a yeast are shown in Fig. 1. The MIC value for E. coli is 4.25 µM, for B. subtilis 2.12 µM and for S. cerevisiae 4.25 µM. The bacterial and yeast cells were treated with SN2 in final concentrations of 2×, 1×, 1/2×, and 1/4× MIC. After 0, 0.5, 1, 3, 6, and 24 h, aliquots were taken and plated on agar plates. After 24 h incubation, the colonies of bacteria and yeast were counted. For E. coli, it was previously reported that the 2× MIC is bactericidal (Herbel, Schäfer & Wink, 2015); this was also observed in the kinetics analysis in which, after 24 h, no viable cells could be detected. For the bacterial strains, the 1× MIC is bacteriostatic. Further, there is no significant difference in the number of viable cells among the 1/2× MIC, the 1/4× MIC, and the control. For S. cerevisiae, a slight effect between the 1/4× MIC and the control is visible.

Bacteriostatic and fungistatic activity of SN2

The growth of bacteria and yeast cells in the presence of several SN2 concentrations was analyzed by measuring the optical density at 600 nm after 0, 3, 6, 7.5, 9, and 24 h and is shown in Fig. 2. In the log phase of bacterial growth, a clear inhibition by SN2 is visible. Even the 1/8× MIC, which showed no bactericidal effect, appeared as a concentration that inhibits the growth of both gram-negative and gram-positive bacteria. For the yeast, an even lower concentration of 1/32× MIC affects cell growth compared to the control. The 1× MIC completely inhibits growth of all the tested cells. In comparison to the time-kill curves, it is clear that SN2 early shows growth-inhibiting activity even at low concentrations, but it does not kill cells not below a 16-fold higher (bacteria) or 128-fold higher (yeast) concentration.

Salt sensitivity of SN2

To analyze the antimicrobial activity of SN2 under physiological conditions, the activity was tested in several salt concentrations. This assay helps to estimate the potential of SN2 as a pharmaceutical drug, because in the human body, SN2 would be exposed to different salt concentrations. Inhibition of SN2 activity by NaCl and KCl is shown in Fig. 3. For all tests, the 1× MIC of the respective strain was used. NaCl or KCl was added in concentrations between 0 and 300 mM to the SN2 solution. Log-phase bacteria or yeast cells were applied, and growth was measured as optical density at 600 nm. 100% activity is referring to a solution without NaCl or KCl. With increasing concentrations of NaCl and KCl, activity of SN2 declined. NaCl and KCl showed similar effects on the reduction of SN2 activity in E. coli and S. cerevisiae. The SN2 activity against B. subtilis was not blocked by KCl concentrations up to 300 mM and NaCl concentrations up to 100 mM.

Hemolytic and hemagglutinating activity of SN2

To estimate a potential risk of SN2 as a pharmaceutical drug, the susceptibility of mammalian erythrocytes was analyzed. The membrane specificity of an AMP is an essential criterion in developing new antibiotic substances. In order to investigate the potential membrane activity of SN2 on mammalian cells, we performed an erythrocyte hemagglutination and hemolysis assay with sheep erythrocytes. SN2 shows an agglutinating effect in the concentration range of 1-17 µM. The agglutinating effect of 1 µM SN2 is shown in Fig. 4B compared with non-treated erythrocytes (Fig. 4A). Hemolysis can be detected at 17 µM SN2 with approximately 30% erythrocytes being lysed, and at 35 µM with 100% hemolysis (Figs. 4C and 4D). No agglutination and hemolysis were observed for the control sample without SN2.

Effect of SN2 on plant membranes

With regard to the unspecific pore-forming effect of SN2, we analyzed the activity of this AMP towards plant cells of Nicotiana tabacum, a close relative to tomato, from which the investigated SN2 originates. Protoplasts were used to study the direct interaction of SN2 with plant membranes. The interaction of SN2 with membranes of the pathogenic mold (F. solani) has been reported earlier and the membrane perforation could be associated with SN2 molecules through trypan staining of cells with perforated biomembranes. In Fig. 5A, the pictures show the cellular alteration of a protoplast at different time points (1–15 min) after addition of SN2 solution. It was observed that the cell membrane rapidly destabilizes at several points and bursts, so that cytoplasmic material can flow out from the cell. Interestingly, the tonoplast (membrane that surrounds the large vacuole) remained stable for 15 min, and the central vacuole was forced out of the shrinking cell. The loss of cytoplasmic material was documented for both protoplasts and undigested suspension cells (Figs. 5B and 5C). The arrows point out the escaping cytoplasm after 5 min following addition of SN2 solution, indicating that SN2 can diffuse through the plant cell wall, seemingly without problems, and act at the cell membrane.