Antimicrobial Activity of Brilacidin against E. coli

Brilacidin (BRI) is a potent small-molecule mimic of antimicrobial peptides that has demonstrated efficacy against various pathogens. To better understand its antimicrobial mechanism of action, we investigated the interactions of BRI with the membranes of Escherichia coli. Specifically, the surface-sensitive nonlinear optical technique, second harmonic laser scattering (SHS), was used to monitor BRI-induced changes in the permeabilities of the dual phospholipid membranes of E. coli by measuring the adsorption and transport of a membrane-permeable indicator molecule (the quaternary ammonium cation, malachite green). Following exposure to 0.6 to 12x MIC BRI, the permeability of the outer membrane was shown to increase 2-fold. Conversely, the permeability of the inner membrane was shown to exhibit more complicated behavior: Permeability increased for near-MIC doses (<= 3x MIC) of BRI but was significantly reduced for higher concentrations. The latter is consistent with the depolarization of the inner membrane, which was previously predicted in a computational study. The interpretation of the SHS results was corroborated with complementary assays that measure the permeability of small polar molecules to periplasmic versus cytosolic enzymes, as well as ATP luminescence and Western blot assays (for accumulation of secreted protein precursors). It is suggested that the mechanism of action of BRI against E. coli consists of a sequential and complementary attack on both membranes, beginning with an immediate and persistent increase in the permeability of the outer membrane and followed by likely depolarization of the inner membrane. This study also illustrates a new quantitative capability for monitoring antimicrobial interactions by using time-resolved SHS.