Bacterial Membranes Permeability Assay

To examine an ability of the peptides to affect barrier function of outer and inner membranes of Gram-negative bacteria, a colorimetric assay with chromogenic markers nitrocefin (Calbiochem-Novabiochem) and o-nitrophenyl-b-D-galactopyranoside (ONPG, AppliChem) and E. coli ML-35p strain was performed as previously described (Shamova et al., 2009; Panteleev et al., 2018). Briefly, the final concentration of E. coli ML-35p cells was of 2.5 × 10⁷ CFU/ml. The concentrations of ONPG and nitrocefin were of 2.5 mM and 20 mM, respectively. Peptides were serially diluted in a 96-well plate with a non-binding surface (NBS, Corning #3641), and the optical density (OD) of the solution rising due to the appearance of the hydrolyzed nitrocefin or ONPG was measured at 492 and 405 nm, respectively the microplate reader AF2200 (Eppendorf). The assay was performed in phosphate buffered saline (PBS) at 37°C under stirring at 500 rpm. Control experiments were performed under the same conditions without the addition of a peptide. The optical absorption of the solution after incubation with melittin for 2 h was taken as 100%. The absorbance of control wells containing no peptides was subtracted from the absorbance value of each well. Three independent experiments were performed, and the curve patterns were similar for all three series.