Figure 1.
Bactericidal activities of four antimicrobial peptides against B. pseudomallei.
Bacterial suspensions were incubated with 5(black), 10(dark grey) and 20(light grey) µM of cationic antimicrobial peptides or CAZ for 1 h. The viability of bacterial cells was determined by a plate counting technique and the data are presented as the mean and the standard deviation of two independent experiments performed in triplicate.
Figure 2.
Long-term killing kinetics of four antimicrobial peptides against B. pseudomallei.
Bacterial suspensions were incubated with bactenecin (2A), BMAP-18 (2B), CA-MA (2C), RTA3 (2D), or CAZ (2E) at concentrations of 0 (black square), 20 (black circle), 50 (black triangle), 100 (black diamond) µM and samples were taken at 1, 2, 3, 4, 5, 6, and 24 h. Colonies were counted and the bactericidal effects (dashed line) were defined as a ≥3-log reduction in colony-forming units (CFU)/ml compared to the initial inoculum. Data are the mean of two independent experiments performed in duplicate.
Figure 3.
Dose and time dependent anti-biofilm activity of four antimicrobial peptides.
Bacterial suspensions at 1×106 CFU/ml were incubated with 20, 50 and 100 µM peptides for 1 (black), 2 (dark grey), 3 (light grey) and 4 (white) hrs at 37°C. The cells were collected and cultured for 2 days and biofilm mass was measured. The anti-biofilm activities were calculated by [(1-AT/AC)×100%], where AT is the absorbance of the biofilm mass from B. pseudomallei treated with the peptides or CAZ, and AC is the absorbance (550 nm) of biofilm mass from bacteria only. Data are the mean of two independent experiments performed in triplicate.
Figure 4.
LPS binding and membrane permeabilization of four peptides on living bacterial cells.
(A) LPS binding of peptides measured with a polymyxin B-BY displacement assay. B. pseudomallei suspensions at 1×106 CFU/ml were incubated with PMB-BY for 1 hour and treated with the peptides at indicated concentrations. Fluorescence of PMB-BY was monitored 30 min after adding peptides. (B) Permeabilization of the outer membrane by the peptides was observed by NPN uptake. B. pseudomallei cell suspension was incubated with NPN in the presence of 50 µM peptide, and NPN fluorescence was monitored at ex/em 350/429 nm at 0–60 min. The fluorescence intensity upon peptide treatment is reported as a percentage of the maximum fluorescence intensity upon TTX-100 treatment. (C) Permeabilization of the inner membrane by the peptides was assayed by ONPG hydrolysis. The E. coli MG 1655 cell suspension was incubated with 50 µM peptides. Activity of leaked β-galactosidase was measured using ONPG as substrate. ONPG hydrolysis was monitored for 0–2 h. Data are the mean of two independent experiments performed in triplicate. Data are the mean of two independent experiments performed in triplicate.
Figure 5.
Peptide binding to model membranes resulted in membrane leakage and altered membrane fluidity.
(A) Peptide binding induced leakage of ANTS/DPX-trapped model membranes was observed. After addition of 20 uM peptides into the liposome suspension, leakage was monitored by measuring an increase in ANTX fluorescence intensity at 530 nm, with the excitation at 353 nm for 360 sec. (B) hanges in fluidity of model membranes was observed. Fluorescence anisotropy of DPH anchored in LUVs (EYPG/PC 3∶1) was calculated from parallel and perpendicular fluorescence intensity values recorded at excitation 355 and emission at 424 nm, after addition of 5, 10, 20, 25 and 50 uM peptide into 10 mM HEPES buffer pH 7.4.
Figure 6.
Electron micrographs of B. pseudomallei deformation and cytoplasmic leakage caused by four antimicrobial peptides.
Bacterial suspensions at 1×106 CFU/ml were incubated with or without 50 µM of the peptides for 2 h in a 37°C incubator. Untreated bacterial cells had smooth membranes and uniform shape (A) but shrunk when they were treated with CAZ (B). Cell deformation and leakage of cytosol was observed upon peptide treatment (C: bactenecin, D: BMAP-18, E: CA-MA, and F: RTA3). Scale bar represents 500 nm.
Figure 7.
Spatial orientations of four antimicrobial peptides in membrane bilayers calculated with the PPM 2.0 program.
Membrane binding modes were calculated for the predicted α-helical conformations of CA-MA (A), RTA3 (B), and BMAP-18 (C) and for the predicted β-hairpin structure of bactenecin stabilized by the disulfide Cys3-Cys11 in the monomeric form (D) and two dimeric forms: with anti-parallel pairing of the C- and N-terminal β-strands of the monomers (E) and with parallel pairing of the N-terminal β-strands of the monomers (F). Bactenecin dimer with pairing of the N-terminal β-strands showed the deepest insertion into the hydrophobic membrane core amongst the four peptides studied. Peptides are shown as ribbon diagrams colored according to secondary structure (red for α-helix, yellow for β-strand, green for loops); basic residues (Arg, Lys, His) are shown by sticks colored blue; non-polar residues (Phe, Trp, Met, Cys, Leu, Ile, Val) are shown by sticks colored green; the hydrophobic membrane boundary (at the level of the lipid carbonyls) is represented by gray dots. Images were produced using PyMol (http://www.pymol.org/).
Figure 8.
β-hairpin model of bactenecin in the oxidized state.
Cys3-Cys11 disulfide bond in gauche-gauche-gauche conformation is formed in the non hydrogen-bonding pair; the 3-residue β-turn is formed by Val6-Val7-Ile8 residues in αRγRαL conformation [56].
Table 1.
Parameters of spatial orientations in the lipid bilayer of antimicrobial peptides.