LPS binding and membrane permeabilization of four peptides on living bacterial cells.

<p>(A) LPS binding of peptides measured with a polymyxin B-BY displacement assay. <i>B. pseudomallei</i> 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. <i>B. pseudomallei</i> 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 <i>E. coli</i> 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.