Outer membrane permeabilization assays

The membrane permeability of the peptides was determined by using the NPN uptake assay¹⁶. NPN is a hydrophobic fluorescent dye that does not readily permeate the bacterial outer membrane. However, when the membrane integrity is compromised, NPN can enter the cell and bind to the bacterial membrane lipids. This causes the dye to show a strong fluorescence. A. baumannii ATCC19606 and P. aeruginosa PAO1 were grown (OD₆₀₀ = 0.4), centrifuged (10,000 r.p.m. at 4 °C for 10 min), washed and resuspended in buffer (5 mmol l⁻¹ HEPES, 5 mmol l⁻¹ glucose, pH 7.4). NPN solution (4 μl at the working concentration of 10 mmol l⁻¹ after dilution) was added to 100 μl of the bacterial solution in a white 96-well plate. The fluorescence was recorded at λ_ex = 350 nm and λ_em = 420 nm. Aqueous solutions of the peptides (100 μl final volume at their MIC against the strain of interest) were added to a white 96-well plate, and fluorescence was recorded for 45 min after no further increase in fluorescence was observed. All assays were done as three biological replicates. The relative fluorescence values were calculated for the entire course of the experiment using non-linear fitting, and the untreated control (buffer + bacteria + fluorescent dye) as baseline. The following equation was applied to show the percentage difference between the fluorescence of the untreated control (baseline) and the sample: