Fig 1.
Circular dichroism (CD) spectral analysis of bovine NK-lysin peptides.
CD spectra of all four bovine NK-lysin peptides (20 μM final concentrations) were determined in the absence of (A) or presence (B) of liposomes (1 mM final concentration) in 10 mM potassium phosphate buffer solution. CD spectra were measured at room temperature using Jasco J-815 CD spectrometer and measurements were taken every 0.1nm from 250 to 190nm. Average spectra with six accumulations after baseline correction for each of the peptides are shown.
Fig 2.
Hemolytic activities of bovine NK-lysin peptides on cattle erythrocytes.
The cattle RBCs were resuspended in PBS (2.5% final hematocrit) and incubated with 5 μM and 30 μM bovine NK-lysin peptides at 37°C in a humidified atmosphere for 60 min. The release of hemoglobin was measured spectrophotometrically at 405 nm and 570 nm wavelengths. RBCs incubated with PBS or 0.1% (v/v) Triton X-100 served as negative and positive controls, respectively. Means and SEM were calculated from two independent experiments. (* = P<0.007).
Fig 3.
Antimicrobial activity of bovine NK-lysin peptides on M. bovis.
A bovine (428E) or bison (NADC1) isolate of M. bovis was incubated with the indicated concentrations of bovine NK-lysin peptides at 37°C in a humidified atmosphere of 5% CO2 for 60 min. The percentage of viable cells remaining was calculated in comparison to the same isolate incubated under identical conditions with only PBS. Results shown are the means and SEM of three independent experiments.
Fig 4.
Plasma membrane depolarization activity of bovine NK-lysin peptides on M. bovis.
M. bovis was incubated with membrane-potential sensitive cyanine dye DiSC3(5) (0.4 μM final concentration) at 37°C for 30 min followed by addition of KCl (100 mM final concentration). Bovine NK-lysin peptides (5 and 20 μM final concentrations) were added and changes in fluorescence were immediately measured using a fluorescent microplate reader with excitation and emission at 622 nm and 670 nm wavelengths, respectively. Triton X-100 (0.1% (v/v) final concentration) was used as a positive control, while ethanol treated (killed) and untreated M. bovis (in PBS) were used as negative controls. Results of one representative experiment out of three are shown.
Fig 5.
Flow cytometric analysis of M. bovis treated with bovine NK-lysin peptides.
Live and dead M. bovis in PBS, NK1, NK2A, NK2B, NK2C, and ethanol treated samples. M. bovis was incubated with 20 μM of NK-lysin peptides at 37°C for 30 min followed by staining with Syto 9 and PI. X axis indicates live bacteria (Syto 9) with intact membranes and Y axis indicates dead bacteria (PI) with damaged membranes. Results of one representative experiment out of three are shown.
Fig 6.
Confocal laser-scanning microscopic analysis of M. bovis treated with bovine NK-lysin peptides.
Live and dead M. bovis in PBS (A), and NK2A (B) samples. M. bovis was incubated with 20 μM NK2A at 37°C for 30 min, and then stained with PI and DAPI. Dead bacteria with damaged membranes (red) were stained with PI and DAPI while live bacteria were stained only with DAPI. Results of one representative experiment out of three are shown.
Fig 7.
PI uptake of M. bovis following incubation with bovine NK-lysin peptides.
M. bovis was incubated with PI (3 μM final concentration) for 5 min followed by addition of 20 μM (A) and 5 μM (B) final concentrations of NK-lysin peptides and PI fluorescence was immediately measured using a fluorescent microplate reader with excitation and emission at 530 nm and 620 nm wavelengths, respectively. Triton X-100 (1% v/v) was used as a positive control and PBS was used as a negative control. Results shown are the means and SEM of three independent experiments. Symbols *, and ** indicate significant differences in PI uptake compared to the PBS-treated control (** = P<0.001; * = P<0.05).
Fig 8.
NK-lysin-induced damage to M. bovis plasma membrane.
Mycoplasma bovis was incubated with PBS (A: control), 30 μM NK2A (B) or 30 μM NK2C (C) at 37°C for 60 min and processed for TEM. M. bovis incubated with NK-lysin peptides show damaged membranes as evidenced by ghost cells (triangles in panel B) and ruptured membranes (arrow in panel C). Control M. bovis (panel A) did not have any damaged membranes or ghost cells.