Elucidating the mechanism by which synthetic helper peptides sensitize Pseudomonas aeruginosa to multiple antibiotics
Fig 3
D-11 impairs ATP and NADH levels, induces the accumulation of ROS, inhibits efflux pumps and promotes intracellular antibiotic accumulation.
(A) Intracellular ATP levels at different D-11 concentrations. 40 mg/L CCCP was used as a positive control. (B) Resazurin to resorufin reduction by NADH at different D-11 doses. (C) Intracellular NADH concentration after different D-11 doses. (D) Total ROS accumulation in PAO1 treated with D-11. Exogenous addition of NAC (6 mM) diminished the accumulation of ROS induced by 16 μM of D-11. (E) D-11 dosage-related EtBr efflux inhibition. PAO1 cells were incubated with EtBr in HEPES for 30 min at 37 °C then the cells were collected and resuspended in MHB medium containing the indicated concentrations of D-11 or 40mg/L CCCP. Then the fluorescence was monitored for 90 min at 37°C. (F) D-11 antagonizes the effect of the efflux pumps inhibitor PAβN. (G) Intracellular D-11 dosage-related increment of azithromycin. (H) β-Galactosidase activity for the combination of azithromycin and D-11. (I) Relative lacZ mRNA expression levels for D-11 and D-11/azithromycin-treated cells. All the tests were performed in triplicate and all the data are presented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant by Student’s t-test.