Elucidating the mechanism by which synthetic helper peptides sensitize Pseudomonas aeruginosa to multiple antibiotics
Fig 2
D-11 increases membranes permeability, interacts with LPS and phospholipids and dissipates the membrane potential.
(A) D-11 dosage-related outer membrane permeability increment. Polymyxin B at 0.5mg/L was used as a positive control. (B) D-11 dosage-related inner membrane permeability increment. 0.5 mg/L polymyxin B was used as a positive control. (C) SYTO9 and PI live/dead cell staining of PAO1 treated with D-11. P. aeruginosa cells were treated with indicated concentrations of D-11 for 60 min. Cells were washed with PBS buffer and stained with SYTO9 and PI before being inspected with a microscope. (D) D-11 activity in the presence of BSA, PC, PE, PG, CL or LPS, the activity for the synergistic relation D-11/azithromycin in the presence of LPS (E), CL (F), and PG (G). D-11 lost the synergistic effect in the presence of LPS, CL or PG. The MIC of azithromycin was determined by checkerboard microdilution assays in the presence of LPS, CL or PG (0–256 mg/L) with 4 μM of D-11 or not. (H) DiSC3(5) membrane potential measurement for several D-11 dosages. Polymyxin B at 0.5mg/L was used as a positive control. The DiSC3(5) dye was incubated with PAO1 for 30 min followed by self-quenching and stabilization, then indicated concentrations of D-11 and SDS were added. The fluorescence units were monitored for 60 min. (I) PMF disruption measurement. CCCP was used as a positive control. All the tests were performed in triplicate and all the data are presented as mean ± SD. **, P < 0.01; ***, P < 0.001 by Student’s t-test.