Fluorescence Imaging of Bacterial Killing by Antimicrobial Peptide Dendrimer G3KL

We recently discovered that peptide
dendrimers such as G3KL ((KL)8(KKL)4(KKL)2KKL, K = branching l-lysine) exert strong
activity against Gram-negative bacteria including Pseudomonas
aeruginosa, Acinetobacter baumannii, and Escherichia coli. Herein, we report a detailed mechanistic
study using fluorescence labeled analogs bearing fluorescein (G3KL-Fluo) or dansyl (G3KL-Dansyl), which show
a similar bioactivity profile as G3KL. Imaging bacterial
killing by super-resolution stimulated emission depletion (STED) microscopy,
time-lapse imaging, and transmission electron microscopy (TEM) reveals
that the dendrimer localizes at the bacterial membrane, induces membrane
depolarization and permeabilization, and destroys the outer leaflet
and the inner membrane. G3KL accumulates in bacteria
against which it is active; however, it only weakly penetrates into
eukaryotic cells without inducing significant toxicity. G3KL furthermore binds to lipopolysaccharide (LPS) and inhibits the LPS
induced release of TNF-α by macrophages, similarly to polymyxin
B. Taken together, these experiments show that G3KL behaves
as a potent membrane disruptive antimicrobial peptide.